CZ253897A3 - Osteoprotegerin - Google Patents

Abstract

The present invention discloses a secreted polypeptide, termed osteoprotegerin, which is a member of the tumor necrosis factor receptor superfamily and is involved in the regulation of bone metabolism. Also disclosed are nucleic acids encoding osteoprotegerin, polypeptides, recombinant vectors and host cells for expression, antibodies which bind OPG, and pharmaceutical compositions. The polypeptides are used to treat bone diseases characterized by increased resorption such as osteoporosis.

Description

Osteoprotegerin

Technical field

The invention generally relates to polypeptides involved in the regulation of bone metabolism. In particular, the invention relates to a novel polypeptide, referred to as osteoprotegerin, which is a member of the tumor necrosis factor receptor superfamily. The polypeptide is used in the treatment of bone diseases characterized by increased bone loss, for example in the treatment of osteoporosis.

BACKGROUND OF THE INVENTION

Polypeptide growth factors and cytokines are secreted factors which, by specifically binding to discrete surface binding receptors, signal a broad spectrum of changes in growth, differentiation and metabolism of cells. Receptors, as a class of proteins, change their structure and mode of signal transduction. They are characterized by having an extracellular domain that is part of the ligand binding and a cytoplasmic domain that transmits the intracellular signal. Receptor expression patterns precisely determine which cells will respond to a given ligand, while the structure of a given receptor dictates a cell response induced by ligand binding. Receptors have been shown to transmit intracellular signals through their cytoplasmic domains by activation of protein tyrosine or phosphorylation of protein serine / threonine (eg, platelet-derived growth factor receptor (PDGFR)) or

01-1718-97 of transforming growth factor-β receptor-I, by stimulating G-protein activation (e.g. β-adrenergic receptor-I (ΤΘΕβΚ-Ι)) and by modulating association with signal transduction cytoplasmic proteins (eg TNFR-1 and Fas (APO) (Heldin, Cell 80: 213-223 (1995)).

The superfamily of the tumor necrosis factor (TNFR) receptor is a family of type I transmembrane proteins that share a conserved cysteine rich motif that repeats three to six times in the intracellular domain (Smith et al., Cell 76, 953-962 (1994)). Together, these repeats form the ligand binding domains of said receptors (Chen et al., Chemistry 270, 2874-2878 (1995)). The ligands for these receptors are a structurally related family of proteins homologous to TNFα. (Goeddel et al., Cold Spring Harbor Symp. Quart. Biol. 51, 597-609 (1986); Nagata et al., Science 267, 1449-1456 (1995)). TNFα binds to the distinct but closely related receptors, TNFR-1 and TNFR-2. TNFα produces a variety of biological responses in receptor-bearing cells, including proliferation, differentiation, and cytotoxicity and apoptosis (Beutler et al., Ann. Rev. Biochem. 57, 505-518 (1988)).

It is believed that TNFα mediates acute and chronic inflammation of the false response ........ (Beutler et al., Ann. Rev.

Biochem. 57, 505-508 (1988)). Systemic delivery of TNFα induces toxic shock and extensive tissue necrosis. As a result, TNFα may be responsible for severe morbidity and mortality associated with a variety of infectious diseases including sepsis. Mutations in FasL, a ligand for the TNFR-related Fas / APO receptor (Suda et al., Cell 75, 1169-1178 (1993)), have been associated with autoimmunity (Fischer et al., Cell 81,

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935-946 (1995)), while FasL overproduction may be involved in drug-induced hepatitis. Thus, ligands for various TNFR-derived proteins often mediate the serious consequences of many disease states; hence agents that neutralize the activity of these ligands have therapeutic value. Soluble TNFR-1 receptors and antibodies that bind TNFα were tested for their ability to neutralize systemic TNFα (Loetscher et al., Cancer Cells 3 (6), 221-226 (1991)). Recently, a naturally occurring form of secreted TNFR-1 mRNA has been cloned and its product tested for its ability to neutralize TNFα activity in vitro and in vivo (Kohno et al., PNAS USA 87, 8331-8335 (1990)). The ability of this protein to neutralize TNFα suggests that soluble TNF receptors bind and purify TNF and thereby block the cytotoxic effects on TNFR-bearing cells.

It is an object of the invention to identify new members of the TNFR superfamily. It is expected that the new family members will be transmembrane proteins or soluble forms thereof, containing extracellular domains and lacking transmembranes and cytoplasmic domains. A new member of the TNFR superfamily that encodes a secreted protein closely related to TNFR-2 has been identified. Analogously to soluble TNFR-1, a TNFR-2-derived protein can negatively regulate the activity of its ligand and may thus be useful in the treatment of certain human diseases.

SUMMARY OF THE INVENTION

A new member of the tumor necrosis factor (TNFR) superfamily was identified in the rat fetal intestinal cDNA library. Full length cDNA was obtained

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the clone that was sequenced. Expression of rat cDNA in transgenic mice showed marked increases in bone density, especially in long bones, pelvic bones and vertebrae. The polypeptide, encoded by the cDNA, is referred to as osteoprotegerin (OPG) and plays an important role in promoting bone accumulation.

The invention provides nucleic acids encoding a polypeptide having at least one of the biological activities of OPG. The invention also provides nucleic acids that hybridize to nucleic acids that encode the mouse, rat, or human OPG shown in Figures 2B-2C (SEQ ID NO: 120), 9A-9B (SEQ ID NO: 122), and 9C-9D. (SEQ ID NO: 124). Preferably, the OPG is mammalian OPG and more preferably human OPG. The invention also encompasses recombinant OPG expressing vectors and host cells and methods for producing recombinant OPG. Antibodies or fragments thereof that specifically bind the polypeptide are also part of the invention.

Methods for treating bone diseases are also part of the invention. Polypeptides are useful in preventing bone loss and can be used to treat any condition that results in bone loss, such as osteoporosis, hypercalcemia, Paget's disease of bone, and bone loss due to joint rheumatism or osteomyelitis, and the like. antisense or gene therapy using the nucleic acids of the invention. Pharmaceutical compositions comprising OPG nucleic acids and polypeptides are also within the scope of the invention.

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Brief description of the pictures

Giant. 1, FASTA analysis of the new EST LORF. Giant. IA shows the derived amino acid sequence inserted into the human TNFR-2 sequence. Giant. IB shows the profile analysis of the new EST LORF, wherein the figure shows the derived FRI-1 amino acid sequence included in the TNFR-profile. Giant. IC depicts the structure of the TNFR superfamily showing a region that is homogeneous with the new FRI-1.

Giant. 2 depicts the structure and sequence of the rat OPG gene over its full length as a new member of the TNFR superfamily. Giant. 2A shows a map of the pMOB-B1.1 insert. The box indicates the position of the LORF in the cDNA sequence (bold line), the black rectangle indicates the signal peptide, and the gray ellipses indicate the position of repeating cysteine-rich sequences). Giant. 2B and 2C show the nucleic acid and cDNA sequences of rat OPG, wherein the putative signal peptide is underlined and potential N-linked glycosylation sites are indicated in bold, underlined. Giant. D and E show the alignment of the corresponding sequences (Wisconsin GCG Package, Version 8.1) of OPG with other members of the TNFR superfamily, fas (SEQ ID NO: 128); tnfr1 (SEQ ID NO: 129); sfu-t2 (SEQ ID NO: 130); tnfr2 (SEQ ID NO: 131); cd40 (SEQ ID NO: 132); osteo (SEQ ID NO: 133); ngfr (SEQ ID NO: 134); ox40, (SEQ ID NO: 135); 4-1bb (SEQ ID NO: 136).

Giant. 3 is a PepPlot analysis (Winsconsin GCG Package, Version 8.1) of the predicted rat OPG protein sequence, and FIG. 3A schematically shows a rat OPG with hydrophobic (top) and hydrophilic (bottom) amino acids. Giant. 3B shows the amino acid residues that make up the β-structure (top) and the refractive β-structure (bottom) »»

01-1718-97 defined by Chou and Fasman (Adv. Enz. 47, 45-147 (1948)). Giant. 3C shows the degree of willingness to form an α-helix and β-structure (Chou and Fasman, ibid.); curves lying above the horizontal axis 1.00 show the willingness to form an α-helix or β-structure; wherein the structure may be terminated in regions of the protein in which the curves fall below 1.00. Giant. 3D shows residues that form the α-structure (top) or that break the α-structure (bottom). Giant. 3E depicts portions of a protein sequence that is similar to those typically found at the amino terminus of the α- and β-structure (Chou and Fasman, ibid.). Giant. 3F depicts portions of a protein sequence that is typically found at the carboxyl terminus of the α- and β-structure (Chou and Fasman, ibid.). Giant. 3G depicts portions of a protein sequence that is generally folded (Chou and Fasman, ibid.). Giant. 3H shows a helical hydrophobic moment (Eisenberg et al., Proc. Natl. Acad. Sci. USA 81, 140-144 (1984)) at each position in the sequence. Giant. 31 shows the average hydropathy of Kýta and Doolith (J. Mol. Biol. 157, 105-132 (1982)) and Goldman et al. (brief review in Ann. Rev. Biophys. Biophys. Chem. 15, 321-353 (1986)).

Giant. 4 shows mRNA expression patterns for OPG cDNA in human tissues. Northern blots were probed using a 32P-labeled rat cDNA insert (Fig. 4A) or a human cDNA insert (Fig. 4B).

Giant. 5 depicts the generation of transgenic mouse expression of OPG cDNA in hepatocytes. Northern blot expression of the HE-OPG transgene in mouse liver.

Giant. 6 shows bone thickening in OPG transgenic mice. Giant. Figures 6A-6F show control mice; and Figures 6G-6

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6J depict OPG expressing mice; at necropsy, all animals were X-rayed and X-ray images were developed. Giant. Figures 6A-6F show X-ray images of control animals and one transgenic non-expression animal (# 28). In these images, the cortex and the illuminated central bone marrow cavity were clearly distinguishable. In X-ray pictures 6G to 6J, i. OPG expressing animals, the bark was poorly definable and the bone marrow density was increased.

Giant. 7 depicts trabecular bone in OPG transgenic mice. Giant. 7A to 7D represent bone photomicrographs of control animals. Giant. Figures 7A and 7B show images of the femur at low magnification (4x, 10x) (Masson trichrome spot). Tartar resistant acid phosphatase (TRAP) spots demonstrate osteoclasts (see arrows) that resorb both cartilage (Fig. 7C) and trabecular bone (D). It is worth mentioning the flattened appearance of osteoclasts on the trabecular bone. Giant. 7E to 7H are photomicrographs of bone of OPG expressing animals. Giant. Figures 7E and 7F show images of the femur at low magnification (4x, 10x) (Masson's trichrome spot), the clear area is cartilage, the blue area represents bone, and the red area represents cortex.

noted that ....... unlike the control trabecular bone did not resorb, resulting in the usual marrow cavities. The resulting trabecula has a diverse appearance with blue and clear areas. Clear areas depict remnants of disc cartilage that have never been remodeled. Based on TRAP staining, these animals were found to have osteoclasts (see arrow) on the growing plate (Fig. 7G), which can reduce their number. However, the trabecular surfaces are oriented away from the growth

You need samples to be absent

In fact, platelets are osteoclast-free (Fig. 7H) and this finding is in direct contrast to control animals (see Fig. 7D).

Giant. 8. Defective monocyte-macrophage development was not detected in HE-OPG expressors. The cause of osteopetrosis in mice is the production of defective M-CSF caused by local mutation in the M-CSF gene. This causes a considerable deficit in circulating macrophages and tissue macrophages. Peripheral blood of OPG expressors contained monocytes as shown by H1E analysis. Immunohistochemistry using F480 antibodies that recognize cell surface antigen on mouse macrophages was used to confirm the presence of tissue macrophages. Giant. 8A and 80 show photomicrographs (4x magnification) of spleens of normal and CR1 overexpressors. It should be noted that both animals have a significant number of F480 positive cells. Monocyte-macrophages were also present in the pulp of normal (Fig. 8B) and HE-OPG overexpressors (Fig. 8D) (40x).

Giant. 9 shows the structure and sequence of murine and human OPG cDNA clones (FIG. 9A, FIG. 9B). Mouse cDNA and protein sequence (Fig. 9C, Fig. 9D), human cDNA and protein sequence. The putative signal peptides are underlined and the possible N-linked glycosylation sites are shown in bold (Fig. 9E, Fig. 9F). The figure also shows sequence alignment and alignment of rat, mouse and human OPG amino acid sequences.

Giant. 10 depicts alignment of Preserved Sequences in the Extracellular Domain of TNFR-1 and Human OPG. The PrettyPlot (Wisconsin GCG Package, version 8.1) alignment of TNFR1 and OPG sequences is described in Example 6. The top line shows the human TNFR1 sequences encoding domains 1-4.

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Human OPG sequences encoding domains 1-4. Preserved residues are highlighted in boxes.

Giant. 11 depicts the three-dimensional structure of human OPG. Side view "Molecule of putative three-dimensional structure of human OPG residues 25-163 (wide line) co-crystallized with human TNF3 (thin line). The orientation (from N-terminal to C-terminal) is indicated by the broad arrows along the OPG polypeptide backbone. The sites of the individual cysteine residue side chains are inserted into the polypeptide backbone to help define separate cysteine-rich domains. The TNFP molecule is sequenced as described by Benner et al. (1993).

Giant. 12 depicts the structure of the OPG cysteine-rich domains. The alignment of the human (upper row SEQ ID NO: 136) and mouse (lower row) OPG amino acid sequences shows the putative OPG domain structure. The polypeptide was divided into two halves; N-terminus (Fig. 12A) and C-terminus (Fig. 12B). It is believed that the N-terminal half contains four cysteine-rich domains (designated 1-4). Intended disulfide bonds within the chain are indicated by bold lines and designated as "SS1," SS2, or "SS3." It is further believed that tyrosine 28 and histidine 75 (underlined) form an ionic interaction. Those amino acids that are believed to interact with the OPG ligand are indicated by dots above the corresponding residue. Cysteine residues found in the C-terminal half of OPG are indicated by boxes.

Giant. 13 depicts expression and secretion of full-length and engineered murine OPG-Fc fusion proteins. Giant. 13A

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01-1718-97 shows a map indicating the fusion points to the human IgG1 Fc domain, which are indicated by arrows. Giant. 13B shows the silver staining of an SDS-polyacrylamide gel of conditioned medium obtained from cells expressing F1.Fc expression vectors (full length OPG fused to Fc on Leucine 401) or CT.Fc (C-terminal modified OPG fused to Fc on threonine 180) fusion protein. The first line shows the parent cell line of the pCEP4 expression vector; the second line shows the cell line F1.Fc of the vector; the third line shows the CT.Fc vector cell line. Giant. 13C shows a western blot of conditioned media obtained from Fl.Fc and CT.Fc fusion protein expression vectors probed with the Fc domain of anti-human IgG1 (Pierce). The first line shows the results for the parent pCEP4 expression vector cell line; the second line shows the results for the cell line F1.Fc of the vector; and the third line shows the results for the CT.Fc vector cell line.

Giant. 14 depicts the expression of human OPG in E. coli. Giant. 14A depicts the construction of a bacterial expression vector. The LORF of the human OPG gene was amplified by PCR and subsequently ligated to an oligonucleotide linker fragment (the upper strand is SEQ ID NO: 137; the lower strand is SEQ ID NO: 127) and ligated into pAMG21 vector DNA. The resulting vector η e schooen exorimize OPG from 32-401 bound to

N-terminal methionine residue. Giant. 14B shows SDS-PAGE analysis of uninduced and environment favorable to induced pAMG21-human plasmid. The first line shows MW standards;

bacterial

OPG 32-401 second line indicates uninduced bacteria; the third line indicates induction at 30 ° C; the fourth line indicates induction at 37 ° C; the fifth line indicates the whole cell lysate at 37 ° C; the sixth line indicates the soluble fraction of the whole cell lysate; seventh ·· · ♦ ··

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row indicates insoluble fraction of whole cell lysate; and the eighth row indicates purified inclusion bodies obtained from whole cell lysate.

Giant. 15 shows analysis of recombinant mouse OPG produced in CHO cells by SDS-PAGE and western blot. In both cases, the same amount of CHO conditioned medium was used, using a reducing sample buffer (left column) or a non-reducing sample buffer (right column). After electrophoresis, the dissolved proteins were transfected onto a nylon membrane and subsequently probed with anti-OPG antibodies. The relative positions of the 55 kd monomeric and 100 kd dimeric forms of OPG are indicated by arrows.

Giant. 16 shows a pulse-chase analysis of recombinant mouse OPG produced in CHO cells. CHO cells were labeled by pulsing with ³⁵ S-methionine / cysteine and subsequently screened for a predetermined time. Metabolically labeled cultures were split into both conditioned media and cells that were purified and subsequently immunologically precipitated with anti-OPG antibodies, and both were used to prepare detergent extracts. Immunologically obtained precipitates were dissolved by SDS-PAGE and exposed to film. The upper left and right rectangles show the results obtained by "analyzing" samples "under non-reducing" conditions. The lower left and right rectangles show the results obtained when analyzing samples under reducing conditions; wherein the left rectangles give results for cell extracts, while the right rectangles give results for conditioned media extracts. The relative mobility of the 55 kd monomeric and 100 kd dimeric forms of OPG are indicated by arrows.

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Giant. 17 depicts the expression of OPG in a CTLL-2 cell line. Serum-free conditioned medium was prepared from CTLL-2 cells and CHO-mu OPG [1-401] transfected cells, which was then concentrated and analyzed by non-reducing SDS-PAGE and western blotting. The left bar indicates CTLL-2 conditioned media. The right bar indicates CHO-muOPG conditioned medium. The relative mobility of the 55 kd monomeric and 100 kd dimeric forms of OPG are indicated by arrows.

Giant. 18 depicts the detection of OPG expression in serum samples and liver extracts obtained from control and OPG transgenic mice. Transgenic mouse samples were prepared as described in Example 4. OPG expression was visualized after SDS-PAGE followed by western blotting using anti-OPG antibodies.

Giant. 19 shows the effects of huOPG [22-401] -Fc fusion protein in vitro on osteoclast formation. The osteoclast formation assay is performed as described in Example 11A in the absence (control) or presence of indicated amounts of huOPG [22-401] -Fc fusion. Osteoclast formation was visualized by histochemical staining of tartrate resistant acid phosphatase (TRAP). Giant. 19A shows OPG added to 100 ng / ml. Giant. 19D shows OPG added to 0.1 ng / ml. Giant. 19E shows OPG added to 0.01 ng / ml. Giant. 19F shows OPG added 'to 0.001 ng / ml. Giant. 19G shows a control sample without the addition of OPG.

Giant. 20 depicts a decrease in TRAP activity of an osteoclast culture that occurs along with increasing amounts of OPG. In the osteoclast formation assay, the indicated concentrations of huOPG [22-401] -Fc fusion protein were used and TRAP activity was quantified as described in Example 11A.

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Giant. 21 shows the effect of OPG on the final stage of osteoclast differentiation. The HuOPG [22-401] -Fc fusion was added in the osteoclast formation assay during the middle stage of osteoclast maturation (days 5 to 6) or during the final stage of osteoclast maturation (days 7 to 15; CTL-OPG). TRAP activity was quantified and compared to that observed in the absence of OPG (CTL-CTL) in the presence of OPG in the whole assay (OPG-OPG).

Giant. 22 shows the effects of IL-β, IL-β, and OPG on blood ionized calcium in the body of mice. Blood ionized calcium levels were monitored following injection of IL-1β alone, IL-1α alone, IL-1β plus muOPG [22-401] -Fc, IL-1α plus MuOPG [22-401] -Fc, and muOPG alone [22- 401] -Fc. Control mice received only phosphate buffered saline (PBS) injections. The IL-ββ experiment is shown in Figure 22A. Experiment II-1a is shown in Figure 22B.

Giant. 23 osteoclasts Histologically depicts the effects of OPG on calvary in control and IL1-treated mice.

methods for analyzing murine calvary bone samples are described in Example 11B. The arrows indicate the osteoclasts contained in the mouse sample after two days of treatment. Giant. 23A depicts calvary samples of mice injected four times with PBS each day. Giant. 23B depicts calvary samples of mice injected daily with one IL-1 injection and three PBS injections. Giant. Fig. 23C shows calvary samples of mice injected daily with one injection of PBS and three injections of OPG; and Fig. 23D shows calvary samples of mice injected daily with one injection of IL-1 and three injections of OPG.

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Giant. 24 shows radiographic analysis of bone accumulation in the marrow cavity of normal mice. Mice were injected subcutaneously with saline (Figure 24A) or muOPG [22-401] -Fc fusion (5mg / kg / day) for fourteen days (Figure 24B) and bone density was determined as described in Example 11C.

Giant. 25 shows a histomorphometric analysis of bone accumulation in the marrow cavity of normal mice. Injection and histological assays were performed as described in Example 11C.

Giant. 26 shows a histological analysis of bone accumulation in the marrow cavity of normal mice. Injection and histological assays were performed as described in Example 11C. Giant. 26A shows injection of saline and FIG. 26B shows injection of muOPG [22-401] -Fc fusion.

Giant. 27 depicts OPG activity administered to ovariectomized rats. This one-week experiment showed that OPG tends to block bone thinning and also showed other anti-resorptive therapies. DEXA measurements were taken at the time of ovariectomy and one and two weeks after treatment. The results were expressed as% change in the initial " bone " -density - (mean-value - *. / - SEM) -, - =

Giant. 28 depicts bone density in femoral metaphysis, as measured by histomorphometric methods, which was found to be lower in ovariectomized rats (OVX) than in animals that were only pretended (SHAM) 17 days after ovariectomy. This effect was blocked by OPG-Fc, with OPG-Fc treated ovarian-depleted rats (OVX + OPG) having significantly higher bone density than rats.

01-1718-97 Ovary-free and vehicle-treated (OVX) (mean +/- SEM).

A new member of the tumor necrosis factor receptor (TNFR) receptor superfamily has been identified as expressed sequence termination (EST), isolated from the intestinal rat fetal cDNA library. The structures of the full length rat cDNA clones and the corresponding mouse and human cDNA clones were determined and described in Examples 1 and 6. The rat, mouse and human genes are shown in Figures 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO). NO: 122), respectively. 9C to 9D (SEQ ID NO: 124). All three sequences showed high similarity to the extracellular domains of TNFR family members. None of the isolated full-length cDNA clones encoded transmembrane and cytoplasmic domains, as would be expected for membrane binding receptors, suggesting that these cDNAs encode soluble secreted proteins rather than cell surface receptors. A portion of nucleotides 1200-1353 constituting the human gene shown in Figure 9D was deposited in the Genbank database on November 22, 1995 under accession number 17188769.

Example 2 describes the observed tissue distribution of rat and human mRNA. mRNA expression in rats was detected at

J VA VΊ and / - «on expression was found in the kidney. Expression in skeletal muscle and pancreas was also detected, expression in the same tissues was detected

In humans it was and also in the lymph nodes, thymus, spleen and appendix.

Rat cDNA was expressed in transgenic mice (Example 3) using a liver specific ApoE promoter expression system. Expression analysis showed •

01-1718-97 Significant increase in bone density, especially in long bones (femur), vertebrae and flat bones (pelvis). Histological analysis of the stained bone portions showed severe osteopetrosis (see Example 4), suggesting a significant ^J imbalance between bone formation and resorption, leading to significant bone and cartilage accumulation. The reduction in the number of trabecular osteoclasts in the bones of OPG expressor animals suggests that a substantial part of TNFR-related protein activity may be represented by protection from bone resorption, an osteoclast-mediated process.

In terms of activity in transgene expressors, TNFR-related proteins, designated OPG, are described herein.

Using a rat cDNA sequence, a mouse and human cDNA clone was isolated (Example 5). The expression of mouse OPG in 2 93 cells and human OPG in E. coli is described in Examples 7 and 8. Mouse OPG was produced as an Fc fusion that was purified by affinity chromatography (protein A). Example 7 also describes the expression of full-length and truncated human and mouse OPG polypeptides in CHO and 293 cells, either as expression of fusion polypeptides on the human IgG1 Fc region or as expression of non-fused polypeptides. Expression of full-length and truncated human and mouse OPG in E. coli either as Fc fusion polypeptides or as non-fused polypeptides is described in Example 8. Purification of recombinantly produced mammalian and bacterial OPG is described in Example 10.

OPG biological activity was determined using an in vitro osteoclast maturation assay, an hypercalcemia-induced interleukin-1 (IL-1) model in vivo, and bone density injection studies in normal mice (see Example 11). The following OPG recombinant proteins produced in CHO

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or 293 cells demonstrated activity in E. coli in the osteoclast maturation assay: muOPG [22-185] -Fc, muOPG [22194] -Fc, muOPG [22-401] -Fc, muOPG [22-401], huOPG [22- 201] Fc, huOPG [22-401] -Fc. muOPG [22-180] -Fc produced in GHO cells and huOPG met [32-401] produced in E. coli did not demonstrate activity in the in vitro assay.

OPG, obtained from several sources, was produced as a dimer and to some extent as a higher multimer. Rat OPG [22401] produced in transgenic mice, muOPG [22-401] and huOPG [22-401] produced as recombinant polypeptide in CHO cells, and OPG expressed as a naturally occurring product of cytotoxic T cell line were predominantly dimers and trimers, as shown by analysis on non-reducing SDS gels (see Example 9). Truncated OPG polypeptides having deletions in the amino acid region 186-401 (eg, OPG [1-185] and OPG [1-194]) were predominantly monomeric, suggesting that the 186-401 region may be part of the self-linkage of OPG polypeptides. However, huOPG met [32-401], produced in E. coli, was largely monomeric.

OPG may be important in the regulation of bone resorption. The protein appears to act as a soluble receptor for the TNF family and may inhibit the interaction between the receptor and the ligand involved in the osteolytic pathway. One aspect of regulation seems to be the reduction of osteokiasts .......

Nucleic acids

The invention provides an isolated nucleic acid encoding a polypeptide having at least one biological OPG activity. As noted herein, biological activities of OPG

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include, but are not limited to, all activities involving bone metabolism, and in particular bone thickening. The nucleic acids of the invention are selected from the following list of acids:

a) the nucleic acid sequences shown in FIGS. 2B to 2C (SEQ ID NO: 120), FIGS. 9A to 9B (SEQ ID NO: 122), respectively; 9C to 9D (SEQ ID NO: 124) or their complementary chains;

b) nucleic acids that hybridize under stringent conditions to the polypeptide-coding region shown in FIGS. 2B to 2C (SEQ ID NO: 120), FIGS. 9A to 9B (SEQ ID NO: 122), respectively. Figures 9C to 9D (SEQ ID NO: 124);

(c) nucleic acids which: hybridize as strict conditions with nucleotides 148 to 337 incl those which Fig. IA; and d) nucleic acid sequences acids, which make up degenerate forms of sequences (a) and (b). The invention provides nucleic acids acids, which code

rat, murine and human OPG as well as nucleic sequences hybridizing to sequences that encode a polypeptide having at least one biological OPG activity. The invention also provides nucleic acids that hybridize to rat OPG EST comprising nucleotides 148-337, as shown in Figure IA. Hybridization conditions are typically high stringency, for example, 5xSSC, 50% formamide, and 42 ° C, as described in Example 1. Equivalent stringency to these conditions can be readily obtained by adjusting the salt and organic solvent concentrations and adjusting the temperature. The nucleic acids in (b) include sequences encoding OPG-derived polypeptides that do not undergo detectable hybridization with other known TNF receptor superfamily members. In a preferred embodiment, the nucleic acids are acids,

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2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122), respectively. 9C to 9D (SEQ ID NO: 124).

The length of the hybridizing nucleic acids of the invention may vary because the hybridization may take place in part of the polypeptide-coding regions or in all of the polypeptide-coding regions shown in Figures 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ. ID NO: 122), respectively. 9C to 9D (SEQ ID NO: 124) and may also occur in adjacent non-coding regions. Thus, hybridizing nucleic acids may represent truncation or extension of the sequences depicted in FIGS. 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122), respectively. 9C to 9D (SEQ ID NO: 124). Truncated or expanded nucleic acids are part of the invention as long as they retain one or more biological properties of OPG. Hybridizing nucleic acids may also include adjacent non-coding regions that are 5 'and / or 3' relative to the OPG coding region. Non-coding regions include regulatory regions that are involved in OPG expression, for example, promoters, enhancers, translation initiation sites, transcription termination sites, and the like.

Hybridization conditions for nucleic acids are described by Sambrook et al. in Molecular Cloning: A Laboratory Manual, 2nd Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

DNA encoding rat OPG was provided in plasmid pMO-B1.1, deposited with the American Type Culture Collection, Rockville, MD, on December 27, 1995, under ATCC accession number 69970. Mouse OPG-encoding DNA was provided in the form of plasmid pRcCMV- OPG mice deposited with the American Type Culture Collection, Rockville, MD,

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27 December 1995, under accession number 69971. Human OPG-encoding DNA was provided in the form of plasmid pRcCMV-human OPG and deposited at the American Type Culture Collection, Rockville, MD, on December 27, 1995, under accession number 69969. Nucleic acids of the invention will hybridize under stringent conditions to the DNA inserts deposited under ATCC Accession Nos. 69969, 69970, and 69971, and have at least one biological OPG activity.

The invention also provides derivatives of the nucleic acid sequences shown in Figures 2B, 9A and 9B. As used herein, derivatives include nucleic acid sequences having additions, substitutions, inserts, or deletions of one or more residues, such that the resulting sequences encode polypeptides having one or more amino acid residues that have been added, omitted, inserted, or substituted, and the resulting polypeptide has OPG activity . Nucleic acid derivatives may be naturally occurring acids, such as splice variants or polymorphisms, or may be constructed using site-directed mutagenesis techniques available to those skilled in the art. An example of a naturally occurring OPG variant is a nucleic acid encoding a change in lys to asn at the 3rd residue of the leader sequence (see Example 5). Nucleic acid derivatives are expected to encode amino acid changes in regions of the molecule that are least likely to interfere with biological activity. Other derivatives include a nucleic acid encoding an OPG membrane binding form having an extracellular domain as shown in Figures 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122), respectively. 9C to 9D (SEQ ID NO: 124) together with the transmembrane and cytoplasmic domains.

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In one embodiment, OPG derivatives comprise nucleic acids encoding truncated forms of OPG from whose carboxyl terminus one or more amino acids have been deleted. Nucleic acids encoding OPG may lack 1 to 216 amino acids at the carboxyl terminus. The Fc region may optionally extend from the new carboxyl terminus to provide a biologically active OPG-Fc fusion polypeptide (see Example 11). In preferred embodiments, the nucleic acids encode OPG having an amino acid sequence identical to residues 22-185, 22-189, 22-194 or 22-203 (using the numbering shown in Figures 9E-9F) and optionally encoding the human IgG Fc region.

Also included are nucleic acids encoding truncated forms of OPG that lack one or more amino acids at the N-terminus. Truncated forms include forms lacking a portion of 21 amino acids or all 21 amino acids containing a leader sequence. In addition, the invention provides nucleic acids encoding OPG that lack 1 to 10 amino acids at the mature N-terminus (at the

And which optionally lacks 1 to 216 amino acids at the C-terminus (at the 401th residue). The nucleic acids may optionally encode a methionine residue at the N-terminus. Examples of such OPG truncated polypeptides are described in Example 8.

Examples of nucleic acids of the invention include cDNA, genomic DNA, synthetic DNA and RNA. cDNA was obtained from libraries prepared from mRNA isolated from various OPG expressing tissues. In humans, OPG's tissue sources are kidney, liver, placenta and heart. Genomic DNA, coding for

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OPG was obtained from genomic libraries that are commercially available for a variety of species. Synthetic DNA was obtained by chemical synthesis of overlapping oligonucleotide fragments, followed by assembly of the fragments into the reconstituted portion of the coding region or all of the coding region and flanking sequences (see U.S. Patent 4,695,623, which describes chemical synthesis of interferon genes). RNA was most readily obtained using prokaryotic expression vectors that direct high-level mRNA synthesis, for example, using vectors using T7 promoters and RNA polymerase.

The nucleic acid sequences of the invention are used for deletion of OPG sequences in biological samples in determining cells and tissues that express OPG mRNA. The sequences can also be used to search for OPG-related sequences in cDNA and genomic libraries. Such screening is one of the methods that those of ordinary skill in the art of deleting homologous sequences under suitable hybridization conditions can routinely perform. Nucleic acids are also useful in modulating the expression of OPG levels by antisense or gene therapy. Nucleic acids are also used in the development of transgenic animals that can be used in polypeptide production and biological activity studies (see Example 3). ~ .... ______

Vectors and host cells

Also included within the scope of the invention are expression vectors comprising OPG encoding nucleic acid sequences, host cells transformed with said vectors, and methods for preparing OPG. Overview of recombinant expression

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proteins can be found in Methods of Enzymology vol. 185, Goeddel, D.V. ed. Academic Press (1990).

Host cells for the preparation of OPG include prokaryotic host cells, for example E. coli, yeast, plant, insect and mammalian host cells. For example, bacterial strains of E. coli HB101 or JM101 are suitable for expression. Preferred mammalian host cells include COS, CHOd-, 293, CV-1, 3T3, juvenile hamster kidney (BHK) cells and others. Mammalian host cells are preferred if post-translational modifications, such as glycosylation and polypeptide processing, are important for OPG activity. Mammalian expression allows the production of secreted polypeptides that can be isolated from the growth medium.

OPG expression vectors contain a minimum number of sequences required to propagate the vector and to express the cloned insert. These sequences include an origin of replication, a selectable marker, a promoter, a ribosome binding site, an enhancer sequence, an RNA splice site, and transcriptional termination sites. Vectors suitable for expression in the aforementioned host cells are readily available. The nucleic acids of the invention are introduced into vectors using standard recombinant DNA techniques. Also included are vectors for tissue-specific expression of OPG.

These vectors include promoters that function in production in mice, specifically in the liver, kidney or other organs, and viral vectors for the expression of OPG in target human cells.

Using a suitable host-vector system, OPG is produced by recombinant culture of a host cell transformed with an expression vector containing the sequences

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of nucleic acids that encode OPG under the conditions under which OPG is produced and by isolating the expression product. OPG is produced in the supernatant of transfected mammalian cells or in inclusion bodies of transformed bacterial host cells. The OPG thus produced can be purified by methods known to those skilled in the art, which will be described below. OPG expression in mammalian and bacterial host systems is described in Examples 7 and 8. Examples of expression vectors for mammalian hosts are plasmids such as pDSRα described in PCT Application No. 90/14363. Examples of expression vectors for bacterial host cells are plasmids pAMG21 and pAMG22-His described in Example 8. The plasmid pAMG21 was deposited with the American Type Culture Collection, Rockville, MD, July 24, 1996 under accession number 98113. The plasmid pAMG22-His was deposited with the American Type Culture Collection, Rockville, MD, Jul. 24, 1996 under accession number 98112. It is understood that the specific plasmids and host cells described herein are illustrative only, and other available plasmids and host cells may be used to express the polypeptides.

The invention also provides for the expression of OPG from endogenous nucleic acids by filtration in vivo or ex vivo by recombination, which allows modulation of OPG = from the host chromosome. Also included is the expression of OPG, which is based on the introduction of exogenous regulatory sequences (eg, promoters or enhancers) that are capable of directing OPG production from sites encoding endogenous OPG. The invention also encompasses the stimulation of endogenous regulatory sequences capable of directing OPG production (eg, by exposure to transcriptional enhancement factors).

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Polypeptides

The invention provides OPG, a new member of the TNF receptor superfamily that exhibits activity related to bone metabolism. OPG is particularly active in inhibiting bone resorption, increasing bone density. OPG refers to a polypeptide having the amino acid sequence of murine, rat or human OPG or a derivative thereof having at least one of the biological activities of OPG. The amino acid sequences of rat, mouse, and human OPG are shown in Figures 2B to 2C (SEQ ID NO: 121), 9A to 9B (SEQ ID NO: 123), respectively. 9C to 9D (SEQ ID NO: 125). An OPG derivative refers to a polypeptide comprising the addition, deletion, insertion or substitution of one or more amino acids such that the resulting polypeptide has at least one of the biological activities of OPG. The biological activities of OPG include, but are not limited to, activities involving bone metabolism. These polypeptides will preferably have 21 amino acids deleted in the N-terminal leader sequence.

OPG polypeptides within the scope of the invention include rat [1-401], rat [22-180], rat [22-401], rat [22-401] -Fc fusion, rat [1-180] -Fc fusion , mouse [1401], mouse [1-180], mouse [22-401], human [1-401], mouse [22-180], human [22-401], human [22-180], human [ 1-180], human [22-18] -Fc fusion, and human met-32-401. The amino acid numbering is shown in SEQ ID NO: 121 (rat), SEQ ID NO: 123 (mouse) and SEQ ID NO: 125 (human). Also included are polypeptide derivatives having deletions or truncated C-termini such that they lack part or all of the amino acid residues 180-401 of OPG; one or more amino acid changes at residues 180-401; deletion of part or all of the OPG - rich domain

(C-terminal) domains of the rich amino acid changes in

01-1718-97 Cysteine, especially a cysteine-distant deletion; and one or more cysteine-rich domains, particularly in the distant (C-terminal) cysteine-rich domain. In one embodiment, the OPG has 1 to about 10 amino acids in the mature N-terminus (wherein the mature N-terminus is the 22nd residue) and optionally 1 to about 216 amino acids in the C-terminus.

Other OPG polypeptides within the scope of the invention include: human [22-180] -Fc fusion, human [22-201] -Fc fusion, human [22-401] -Fc fusion, mouse [22-185] -Fc fusion , mouse [22-194] -Fc fusion. These polypeptides are produced in mammalian host cells, such as CHO or 293 cells. Other OPG polypeptides of the invention are the following polypeptides that are expressed in prokaryotic host cells: human met [22-401], Fc-human met [22-401] fusion (the Fc region is bound to the N-terminus of full-length OPG) the coding sequence, as described in Example 8), a human met [22-401] -Fc fusion (the Fc region binds to the full-length OPG sequence),

Fc-mouse met [22-401] fusion, mouse met [22-401] -Fc fusion, human met [27-401], human met [22-185], human met [22-189], human met [22-401] -194], human met [22-194] (P25A), human met [22-194] (P26A), human met [27-185], human met [27-189], human met [27-194], human met-agr-gly-ser (his) e [22-401], human met-lys [22-401], human met (lys) ₃ - [22-401], human met [22-401] -Fc (P25A), human met [22-401] (P25A), human met [22-401] (P26A), human met [22-401] (P26D), mouse met [22-401], mouse met [27- 401], mouse met [32-401], mouse met [27-180], mouse met [22-189], mouse met [22-194], mouse met [27-189], mouse met [27-194] , mouse metlys [22-401], mouse HEK [22-401] (A45T), mouse met-lys • ·

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(his) 7 [22-401], mouse met-lys [27-401] - (his) _7, and mouse met [27401] (P33E, G36S, A45P). It will be appreciated that the above OPG polypeptides produced in prokaryotic host cells have an N-terminal methionine residue unless the residue is directly labeled. In specific examples, the 227 amino acid region of human IgG1-γΙ, which had the sequence given by Ellison et al., Was used to produce OPG-Fc fusion. in (Nuc. Acids Res. 10, 4071-4079 (1998)). However, other variants of the human IgG Fc region may also be used.

Analysis of the biological activity of the C-terminal OPG splices condensed with the human IgG1 Fc region indicates the portion of the OPG of about 164 amino acids that is required for the activity. This region comprises amino acids 22-185, preferably the amino acids shown in Figures 9C to 9D (SEQ ID NO: 125), and contains four cysteine rich domains that are characteristic of the cysteine rich TNFR domains of extracellular domains.

Using homology between OPG and extracellular ligand-binding domains of TNF receptor family members, a three-dimensional OPG model was produced based on the known crystalline structure of the extracellular domain of TNFR-I (see Example 6). This model was used to identify those residues in OPG that might be important for biological activity. Cysteine residues have been identified that are involved in maintaining the structure of the four cysteine-rich domains. The following disulfide bonds have been identified in the model: domain 1: cys41 to cys54, cys44 to cys62, tyr23 and his66 can stabilize the structure of this domain; domain 2: cys65 to cys80, cys83 to cys98, cys87 to cysl05; domain 3: cysl07 to cys188, cys124 to cysl42; Domain 4: cysl45 to cysl60, •

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· 1 · 1 · cysl66 to cysl85. As shown in Figures 11 and 12A to 12B, residues that were in close proximity to TNFP were also identified. In this model, OPG binds to the corresponding ligand; TNFP is used as a model ligand to simulate the interaction of OPG with its ligand. In this model, the following residues in OPG have been shown to be important for ligand binding: glu34, lys43, pro66 to gln91 (especially pro66, his68, tyr69, tyr70, thr71, asp72, ser73, his76, ser77, asp78, glu79, leu81, tyr82, pro85, val86, lys88, glu90 and gln91), glul53 and ser155.

Changes in these amino acid residues, either alone or in combination, can affect the biological activity of OPG. For example, changes in specific cysteine residues may alter the structure of individual cysteine-rich domains, while changes in residues that are important for ligand binding may affect physical interactions of OPG with ligands. Structural models can assist in identifying analogs having more desirable properties, such as increased biological activity, greater stability, or an easier formulation method.

The invention also provides an OPG multimer comprising OPG monomers. OPG appears to be active as a multimer (e.g., a dimer, a trimer, a multimer containing a higher number of monomers). Preferably, the OPG multimers are dimers or trimers. OPG multimers may comprise monomers having an OPG amino acid sequence sufficient to support multimer formation, or may comprise monomers having heterologous sequences, for example, an Fc region of an antibody. Analysis of the C-terminal deletion of OPG suggests that at least a portion of the 186-401 region is in association with OPG polypeptides. Substitution of part or all of the OPG amino acid region 186-401

9 • «» · 9

9

01-1718-97 An amino acid sequence capable of self-linkage is also within the scope of the invention. Alternatively, OPG polypeptides or derivatives thereof may be modified to form dimers or multimers by site-directed mutagenesis that results in the formation of unpaired cysteine residues for interchain disulfide bond rotation, photochemical crosslinking, e.g., UV light exposure, or chemical crosslinking with bifunctional linkers, e.g. .

Modifications to OPG polypeptides are also contemplated by the invention and include post-translational modifications (e.g., N-linked or O-linked carbohydrate chain, N-terminal or C-terminal processing), attachment of chemical residues to the amino acid backbone, chemical modifications of N -bound or O-linked carbohydrate chain and addition of the N-terminal methionine residue due to the expression of a prokaryotic host cell. Polypetides may also be modified so that they can be detected, for example enzymatically, with a fluorescent substance, an isotope, or by affinity labeling, which allows the protein to be detected and isolated.

Other modifications of OPG include chimeric proteins in which OPG is fused to a heterologous amino acid sequence. The heterologous sequence may be any sequence that allows the resulting fusion protein to retain OPG activity. Heterologous sequences include, for example, immunoglobulin fusions, for example Fc fusions, which can assist in the purification of this protein. A heterologous sequence that promotes the association of OPG monomers into dimers, trimers and other higher multimeric forms is preferred.

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The polypeptides of the invention are isolated and purified from other polypeptides present in tissues, cell lines, and transformed OPG expressing host cells, or are purified from components of cell structures containing the secreted protein. In one embodiment, the polypeptide is released from association with other human proteins as an expression product of a bacterial host cell.

The invention also provides chemically modified OPG derivatives that may provide additional advantages, such as increased stability and circulation time of the polypeptides, or reduced immunogenicity (see US Patent 4,179,337). The chemical moieties for the derivative may be selected from water-soluble polymers such as polyethylene glycol, a copolymer of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Polypeptides may be modified at random positions of the molecule or at predetermined positions of the molecule bonded chemical residues.

The polymer may be a polymer of any molecular weight and may be branched or straight. For polyethylene glycol, a preferred molecular weight is about 1 kDa to 100 kDa for ease of handling and manufacture (the term approximately indicates that in the polyethylene glycol preparations some molecules will be heavier and some lighter than the stated molecular weight). Obviously, polymers of different molecular weight may be used, the size of which will depend on the desired therapeutic profile (e.g., the desired duration of long-term release, effects, biological activity, difficulty of handling, degree or lack of antigenicity, and others).

01-1718-97 Eng · · ··· * · · · · ··············

Known effects of polyethylene glycol on the therapeutic protein or analogue thereof).

When binding polyethylene glycol molecules (or other chemical residues) to a protein, all possible effects on the functional or antigenic domains of the protein should be considered. A variety of possible binding methods are available to those skilled in the art, for example, the method described in EP 0 401 384 (binding of PEG to G-CSF), see also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (describing pegylation of GM-CSF with tresyl chloride). For example, polyethylene glycol can be covalently linked via amino acid residues via a reaction group, for example, via a free amino or carboxyl group. Reaction groups are those groups to which activated polyethylene glycol molecules can be attached. Amino acid residues having a free amino group may include lysine residues and N-terminal amino acid residues; and those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and a C-terminal amino acid residue. Mercapto groups may also be used as the reaction group for binding the polyethylene glycol molecule (s). For therapeutic purposes, attachment to an amino group is preferred, for example attachment to the N-terminus or lysine group. __ __ ₌

Sometimes, a particular N-terminus chemically modified protein may be desirable. Using polyethylene glycol as an illustrative example of the compositions of the invention, polyethylene glycol can be selected from a variety of polyethylene glycol molecules (with different molecular weights, different branches, etc.). Different ratios of polyethylene glycol molecules to protein molecules can also be selected.

Ρ

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(or peptide) in the reaction mixture, the types of pegylation reactions to be performed, and methods of obtaining the selected N-terminally pegylated protein. A method for obtaining an N-terminal pegylated composition (i.e., separating this residue from other monopegylated residues if desired) may also be to purify the N-terminal pegylated material from a population of pegylated protein molecules. Selective N-terminal chemical modification can be accomplished by reductive alkylation which utilizes different reactivity of different types of primary amino groups (lysine versus N-terminus) available for derivatization in a particular protein. For. Suitable reaction conditions can be achieved by using a polymer containing a carbonyl group essentially to selectively derivatize the protein at the N-terminus.

Synthetic OPG dimers can be prepared by a variety of chemical crosslinking methods. The OPG monomers may be chemically coupled in any manner that preserves or enhances the biological activity of OPG. A wide variety of chemical cross-linking agents can be used for this purpose, depending on the desired protein dimer. For example, the crosslinking agents may be short and relatively stiff or longer and more flexible, may be biologically reversible, and may provide reduced immunogenicity or a longer pharmacokinetic half-life. _ __ _

In one example, OPG molecules are coupled via the N-terminus through a two-step synthesis (see Example 12). In the first step, OPG is chemically modified at the N-terminus by introducing a protected thiol group at this end, which, after purification, is deprotected and used as a binding site for site-specific conjugation, where the second OPG molecule can bind across the entire a variety of different

01-1718-97 Crosslinking agents. N-terminal cross-links include, but are not limited to, disulfide linkage, thioether linkages using bis-functional short-chain aliphatic crosslinking agents, and thioether linkages to bifunctional polyethylene glycol crosslinking agents of varying lengths (PEG dumbbells). The PEG dumbbell of the synthesis of OPG dimers also includes a byproduct of this synthesis, referred to as a "monobell." The OPG monobell is a monomer attached to a linear bifunctional PEG via a free polymeric end. Alternatively, OPG can be cross-linked directly through a variety of amine-specific homobifunctional crosslinking techniques, including the use of reagents such as: diethylenetriaminepentaacetic acid dianhydride (DTPA), p-benzoquinone (pBQ) or bis (sulfosuccinimidyl) suberate (BS ³ ), as well as other agents known in the art. It is also possible to attach reagents such as iminothiolane in the presence of various bifunctional thiol-specific crosslinkers such as bismaleinimide to OPG thiolate to achieve dimerization and / or dumbbell formation in a single process step.

The invention also provides a method of purifying OPG from natural sources and transfected host cells. The purification process can be carried out in one or more standard protein purification steps, performed in an order suitable to obtain the purified protein. Chromatographic steps may include ion exchange, gel filtration, hydrophobic interaction, reverse phase, chromatographic focusing, affinity chromatography using an anti-OPG antibody or biotin-streptavidin affinity complex, and the like.

• · · · «

01-1718-97 English • · · ·

Antibodies

Also provided are antibodies specifically binding to OPG. Antigens for generating antibodies may be full length peptides or peptides formed by part of the OPG sequence. Immunological procedures for generating OPG-reactive polyclonal or monoclonal antibodies are known to those skilled in the art (see, for example, Harlow and Lane, Antibodies: Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. (1988)). The antibodies thus produced are characterized by binding specificity and are epitope resolvable by standard enzyme-linked immunosorbent assays. Antibodies also include chimeric antibodies having variable domain regions and constant domain regions derived from different species. In one embodiment, the chimeric antibodies are humanized antibodies having mouse variable domains and a human constant domain. Complementary determination regions grafted onto the human network (so-called CDR-grafted antibodies) are also part of the invention. Chimeric and CDR-grafted antibodies are made by recombinant methods known to those skilled in the art. Also included are human antibodies produced in mice.

The anti-OPG antibodies of the invention can be used as an affinity reagent in the purification of OPG from biological samples (see Example 10). In one method, the antibody is immobilized on CnBr-activated Sepharose and the antibody-Sepharose conjugate column is used to remove OPG from liquid samples. The antibodies are also used as diagnostic reagents for the detection of α

01-1718-97 How to quantify OPG in biological samples when detection and quantification are performed as described below.

Pharmaceutical compositions

The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the polypeptides of the invention together with a pharmaceutically acceptable diluent, carrier, diluent, emulsifier, preservative and / or adjuvant.

The term "therapeutically effective amount" means an amount that provides a therapeutic effect for a specified condition and route of administration. The composition may be in liquid or lyophilized form and comprises a diluent (Tris, acetate or phosphate buffers) having different pH values and ionic strengths, a solvent such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as acid ascorbic or sodium pyrosulfite. Also included within the scope of the invention are compositions comprising OPG modified with water-soluble polymers that increase their solubility or stability. The compositions may also include incorporating OPG into liposomes, microemulsions, micelles or vehicles that allow long-term controlled delivery of the active agent. Specifically, the OPG compositions may comprise incorporation into polymer matrices, for example, hydrogels, silicones, polyethylenes, ethylene-vinyl acetate copolymers, or biodegradable polymers. Useful hydrogels are, for example, polyhydroxyalkyl methacrylates (p-HEMA), polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol and various polyelectrolyte complexes. Biologically an example

01-1718-97 Degradable polymers are, for example, poly (2-hydroxypropanoic acid) (PLA), polyglycolic acid (PGA), PLA and PGA copolymers, polyamides and polymers of polyamides and polyesters. - Other controlled release formulations include microcapsules, microspheres, macromolecular complexes, and polymeric beads that can be injected.

The choice of appropriate composition will depend on a number of factors including the condition being treated, the mode of administration and the desired pharmacokinetic parameters. A more extensive review of ingredients suitable for pharmaceutical compositions can be found in Remington ⁷ with Pharmaceutical Sciences, 18th edition of AR Gennaro, ed. Mack, Easton, PA (1980).

The compositions of the invention may be administered by injection, either by subcutaneous, intravenous or intramuscular, or by oral, nasal, pulmonary or rectal routes. The choice of route of administration may possibly depend on a number of factors and it should not be a problem for the person skilled in the art to choose the most appropriate route of administration in particular cases.

The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of the nucleic acids of the invention together with a pharmaceutically acceptable adjuvant. The nucleic acid compositions will be suitable for delivering a portion of the OPG coding region or all of that region to cells and tissues as part of an antisense or gene therapeutic regimen.

• o

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Method of treatment

Bone tissue forms the body's support and consists of mineral (largely calcium and phosphorus), a matrix of collagen and non-collagen proteins, and cells. There are three types of cells in the bone, ie osteocytes, osteoblasts and osteoclasts, which are involved in the dynamic process by which bone is continuously formed and resorbed. Osteoblasts promote bone formation, while osteoclasts are associated with resorption. Resorption, or dissolution of bone matrix and mineral, is a fast and effective process compared to bone formation and can release large amounts of minerals from bone. Osteoclasts are involved in the regulation of normal skeletal tissue reshaping and hormone-induced resorption. For example, resorption is stimulated by the secretion of parathyroid hormone in response to an increase in the concentration of calcium ions in extracellular fluids. On the other hand, inhibition of resorption is an essential function of calcitonin. In addition, vitamin D metabolites affect bone sensitivity to parathyroid hormone and calcitonin.

Upon maturation of the skeleton, the amount of bone in the skeleton reflects the balance (or imbalance) of bone formation and bone resorption. Maximal bone marrow reaches after the skeleton matures, before the fourth decade. Between the fourth and fifth decades, the equilibrium shifts and bone resorption dominates. The inevitable decrease in bone mass, which occurs with increasing age, occurs earlier in women than in men, and in some women it is significantly accelerated after menopause (especially Europeans and Asians).

Osteopenia is a disease state generally related to any reduction in bone mass below normal levels. This

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the disease state may arise from slowing bone synthesis or accelerating bone destruction, or both. The most common form of osteopenia is primary osteoporosis, also referred to as postmenopausal and senile osteoporosis. This form of osteoporosis is a condition manifested by an overall loss of bone mass that occurs with increasing age and generally results from an increase in bone resorption at a normal rate of bone formation. Approximately 25 to 30% of all white women in the United States have developed symptomatic osteoporosis. There was also a direct relationship between osteoporosis and the incidence of hip fracture, femur, neck fracture and intertrochanteric fracture in women over 45 years of age. In men, symptomatic osteoporosis develops between the ages of 50 and 70, but it has been found that the disease mainly affects women.

The cause of postmenopausal and senile osteoporosis is unknown. Several factors have been identified that are involved in the development of this disease state. These factors include, for example, a change in hormone levels that accompanies aging and inadequate calcium consumption, which contributes to a decrease in intestinal absorption of calcium and other minerals. Treatment typically involves hormone therapy or the administration of dietary supplements to slow the process. However, to date no effective method of treating bone loss has been found.

The invention provides a method for treating bone diseases, comprising administering a therapeutically effective amount of OPG. The bone disease may be any disease characterized by total bone loss (osteopenia or osteolysis). Treatment with OPG is usually

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used when bone resorption needs to be suppressed. Thus, treatment can be used to reduce bone resorption if this resorption exceeds the normal state, or to reduce bone resorption below the normal value, and to compensate for a lower level of bone formation.

Diseases that can be treated with OPG are as follows:

osteoporosis, for example, primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparathryoidism, Cushing's disease and acromegaly), hereditary and congenital forms of osteoporosis (congenital bone fragility, homocystinuria, Menkes syndrome, and Riley-Day syndrome), and osteoporosis;

Paget's disease of bone (deforming bone inflammation) in adults and adolescents;

osteomyelitis or an infectious lesion in the bone leading to bone loss;

hypercalcaemia resulting from solid tumors (breast, lung and kidney) and hematologic malignancies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcaemia and hypercalcemia associated with hyperthryoidism and renal function disorders;

post-operative osteopenia induced by steroid administration and associated with small and large bowel diseases and chronic hepatic and renal diseases;

osteonecrosis, or death of bone cells, associated with traumatic trauma or non-traumatic necrosis associated with Gaucher disease, sickle cell disease, systemic lupus erythematosus and other disease states;

··

01-1718-97 Bone loss due to joint rheumatism;

periodontal bone loss;

osteolytic metastasis.

Obviously, OPG can be used alone or in conjunction with other factors in the treatment of bone diseases. In one embodiment, osteoprotegerin is used in conjunction with a therapeutically effective amount of a bone stimulating factor. These factors include, but are not limited to, bone morphogenic factors designated BMP-1 to BMP12, transforming growth factor-β (TGF-β) and members of the TGF-β family, interleukin-1 inhibitors, TNFα inhibitors, parathyroid hormone and analogs, parathyroid derived protein and analogues thereof, the E series of prostaglandins, bisphosphonates (for example alendronate and others) and co-enriching minerals, for example fluoride and calcium.

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DETAILED DESCRIPTION OF THE INVENTION

Example 1

Identification and isolation of rat OPG cDNA

Materials and methods for cloning and cDNA analysis are described in Maniatis et al., Ibid. Polymerase chain reactions (PCR) were performed using a PerkinElmer 9600 thermocycler and a PCR reaction mixture (Boehringer-Mannheim) and primer concentrations specified by the manufacturer. Generally, 25 to 50 μΐ of the reaction mixture was denatured at 94 ° C, followed by 20 to 40 cycles, during which the mixture was heated to 94 ° C for five seconds, then cooled to 50 to 60 ° C for 5 seconds and re-heated to 3 to 5 minutes at 72 ° C. At the end of the cycles, the mixture was heated to 72 ° C for 3-5 minutes. Thereafter, the reaction mixture was analyzed by gel electrophoresis as described by Maniatis et al., Ibid.

For EST analysis (Adams et al., Science 252, 1651-1656 (1991)), a cDNA library was constructed using mRNA isolated from the gut d20 intestine. The germs of the rats were dissected whole of the small and large intestine at necropsy and washed in PBS. Whole cellular RNA was purified by acid guanidinium thiocyanate-phenol-chloroform extraction ^- (Chomczynski and Sacchi, Anal. Biochem. 162, 56159, (1987)). The (A +) mRNA fraction was obtained from the total RNA preparation by adsorption to Dynabeads Oligo (dT) 25 (Dynal Corp) and eluting from Dynabeads Oligo (dT) 25 (Dynal Corp) using manufacturer's recommended procedures. The random primer cDNA library was prepared using the Superscript Plasmid System (Gibco BLR, Gaithersburg, Md). Random cDNA primer containing an internal restriction site;

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which was used to initiate first-chain synthesis, had the following sequence:

5 '-AAAGGAAGGAAAAAAGCGGCCGCTACANNNNNNNNT-3' (SEQ ID NO: 1) Not I

For the synthesis of the first strand of a separate reaction mixture, the poles of (A) RNA and 120 ng, 360 ng or 1.080 ng of the random primer. After synthesis of the second chain, the reaction products were separately extracted with a mixture of phenol, chloroform and isoamyl alcohol (25: 24: 1 ratio), whereupon ethanol precipitated. The double stranded (ds) cDNA products of the three reaction mixtures were pooled and ligated to the following ds oligonucleotide adapter:

three were prepared containing 2.5 μς

5'-TCGACCCACGCGTCCG-3 '(SEQ ID NO: 2)

3'-GGGTGCGCAGGCp-5 '(SEQ ID NO: 3)

After ligation, cDNA was completely digested with Not I, extracted with phenol, chloroform and isoamyl (25: 24: 1 ratio) to precipitate alcohol and ethanol. The resuspended cDNA was then size fractionated by gel filtration using preformed columns fitted with the Superscript Plasmid System (Gibco BLR, Gaithersburg, Md) in the manner recommended by the manufacturer.

Two fractions containing the largest cDNA products were collected and, after ethanol precipitation, were directly ligated into Not I and Sal I digested with pMOB vector DNA (Strathmann et al., 1991). The ligated cDNA was electroporated into competent ElectroMAX DH10B E. coli (Gibco BLR, Gaithersburg, MD). For the purpose of automatic sequence analysis ♦ φ φ · φ · · φ φ •

Approximately 10,000 transformants were placed in 20 cm x 20 cm agar plates containing ampicillin-enriched LB nutrient medium. The grown colonies were harvested and plated in 96-well microtiter plates containing 200 ml ¹ L-broth, 7.5% glycerol and 50 gg / ml ampicillin. Cultures that were grown overnight at 37 ° C and a duplicate set of microtiter plates prepared using a sterile 96-pin replication tool were stored at -80 ° C for further analysis. When cloning full-length cDNA, approximately one million transformants were plated on 96 bacterial ampicillin plates, each containing approximately 10,000 clones. Plasmid DNA from each well was

separately isolated behind use set Qiagen Plasmid Maxi Kit (Qiagen Corp., Germany) and deployed se do 96 microtiter plates for the purpose of PCR analysis. For sequencing random fetal rats

For intestinal cDNA clones, glycerol arrays were thawed and small aliquots were diluted 1:25 in distilled water. Approximately 3.0 μΐ of diluted bacterial cultures were added to the PCR reaction mixture (BoehringerMannheim) containing the following oligonucleotides:

5'-TGTAAAACGACGGCCAGT-3 '(SEQ ID NO: 4)

5'CAGGAAACAGCTATGACC-3 '(SEQ ID NO: 5)

The reaction mixtures were incubated in a thermocycler (PerkinElmer 9600) under the following cyclic conditions: 94 ° C for 2 minutes; 30 cycles comprising 5 seconds at 94 ° C, 5 seconds at 50 ° C and 3 minutes at 72 ° C; and finally 4 minutes at 72 ° C. After incubation in a thermocycler, the reaction mixtures were diluted with 2.0 mL of water. The amplified DNA fragments were further purified using Centricon columns (Princeton

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Separations) and procedures recommended by the manufacturer. PCR reaction products were sequenced on an Applied Biosystems 373A automated DNA sequencer using T3 primer reactions (oligonucleotide 353-23; 5'-CAATTAACCCTCACTAAAGG-3 ') (SEQ ID NO: 6) Taq dye terminator (Applied Biosystems) performed according to the manufacturer's recommendations .

The resulting 5 'nucleotide sequence, obtained from randomly collected cDNA clones, was translated and subsequently compared to an existing database of known protein sequences using a modified version of the FASTA program (Pearson et al., Meth. Enzymol. 183, (1990)). The translated sequences were also analyzed for the presence of a specific, cysteine-rich protein motif found in all known members of the tumor necrosis factor receptor (TNFR) superfamily (Smith et al., Cell 76, 959-962 (1994)) using a sequence profile. methods of Gribsk et al. (Proc. Natl. Acad. Sci. USA 83, 4355-4359 (1987)), modified by Lueth et al. (Protein Science 3: 139-146 (1994)).

Using data obtained by FASTA and Profile analysis, EST, FRI-1 (fetal rat intestine-1) was identified as a possible new member of the TNFR superfamily. FRI-1 contained an approximately 600 bp insert with an LORF of approximately 150 amino acids. The closest match for TNFR was human type II TNFR (TNFR-2) in the database. The comparison region within this approximately 150 k and to LORF showed approximately 43% homology between TNFR-2 and FRI-1. Profile analysis, using the first and second cysteine-enriched repeats of the TNFR superfamily, yielded an early Z of approximately 8, suggesting that the FRI-1 gene may encode a new family member. In order to determine the deduction of the FRI-1 product structure, it is fetal

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the rat intestinal cDNA library was screened to determine full length clones. The following oligonucleotides were derived from the original FRI-1 sequence:

5 '-GCATTATGACCCAGAAACCGGAC-3' 5 '-AGGTAGCGCCCTTCCTCACATTC-3' (SEQ ID NO: 7) (SEQ ID NO: 8)

These primers were used in PCR reactions to screen 96 wells of plasmid DNA, each well containing plasmid DNA from 10,000 independent cDNA clones. Approximately 1 gg of plasmid well DNA was amplified in a PCR reaction mixture (Boehringer-Mannheim) using a PerkinElmer 96-well thermocycler under the following cyclic conditions: 2 minutes at 94 ° C, one cycle; 15 seconds at 94 ° C, followed by 45 seconds at 65 ° C, 30 cycles; 7 minutes at 65 ° C, 1 cycle. The PCR reaction products were analyzed by gel electrophoresis. 13 of the 96 plasmid DNA wells gave rise to amplified DNA products with the expected relative molecular weight.

DNA from one positive well was used to transform competent ElectroMAX DH10B E. coli (Gibco BRL, Gaithersburg, MD) as described above. Approximately placed on sterile (BA-85, Schleicher and Schuell) a

000 transformants nitrocellulose fibers ___o σ 1 qXo-c

- The IAU -u Cuííc for ^- Used ³² P-dCTP labeled version of the PCR product obtained above. The filters were prehybridized for 2-4 hours at 42 ° C in 5X SSC, 50% deionized formamide, 5X Denhardt's solution, 0.5% SDS, and 100 gg / ml denatured salmon seed DNA. The filters were then hybridized for approximately 18 hours at 42 ° C in 5X SSC, 50% deionized formamide, 2X Denhardt's solution, 0.1% SDS, 100 gg / ml denatured salmon seed

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I * fr 9

9

DNA and approximately 5 ng / ml labeled probe. Then the filters were washed for 10 minutes at room temperature in 2X SSC, for 10 minutes at 55 ° C in 1X SSC and finally for 10-15 minutes at 55 ° C in 0.5X SSC. Hybridization clones were detected by subsequent autoradiography and subsequently transferred to nitrocellulose filters for second screening purposes. After the second screening, a plasmid clone (pB1.1) was isolated and subsequently amplified in L-growth medium containing 100 gg / ml ampicillin to produce plasmid DNA. Both strands of the 2.4 kb pBl.1 insert were sequenced.

FASTA analysis of the public database to which pB1.1 insertion sequence was used was used to detect any existing sequence that would correspond to and / or exhibit some degree of similarity. No sequence was found to correspond to any known gene or EST, but showed approximately 45% similarity to the human and murine TNFR-2 gene. The methionine starting codon was found at bp 124 of the nucleotide sequence, followed by LORF encoding 401 and residues ending at bp 1327. The 401k residue is believed to have a hydrophobic signal peptide of approximately 31 residues at its N-terminus and 4 potential N-linked glycosylation sites. No hydrophobic sequence overlapping the ₌ transmembrane was identified by PepPlot (Wi-sconsin - CGC-package, version 8.1). The putative 401 ak sequence was subsequently used for protein database research. Again, no existing corresponding sequences were found, although there was a strong similarity with many members of the TNFR superfamily, most notably human and murine TNFR-2. Sequence alignment of this novel protein with known members of the TNFR superfamily was prepared using the Pileup a program

01-1718-97 was subsequently modified using PrettyPlot (Wisconsin GCG package, version 8.1). This alignment shows a pure homology between the long FRI-1 gene product and all other members of the TNFR family. The homology region maps the extracellular domain of TNFR family members and corresponds to the three or four cysteine-rich repeats found in the ligand binding domain of these proteins. Thus, the FRI-1 gene encodes a new member of the TNFR family. Since no transmembrane region was detected, it is believed that it may be a secreted receptor similar to soluble TNFR-1-derived receptors (Kohno et al., Proc. Natl. Acad. Sci. USA 87, 8331-8335 (1990)). Due to the apparent biological activity of the FRI-1 gene (see above), the product was designated as osteoprotegerin (OPG).

Example 2

OPG mRNA expression patterns in tissues

To determine the size of the human transcript and expression patterns, multiple Northern blots of human tissue (Clonetech) were probed with a ³² p-dCTP labeled FRI-1 PCR product. Northern blots were prehybridized at 42 ° C for 2-4 hours in 5X SSPE, 50% formamide, 5X Denhardt's solution, 0.5% SDS, and 100 gg / ml denatured salmon seed DNA. The blots were then hybridized for 18-24 hours at 42 ° C in 5X SSPE, 50% formamide, 2X Denhardt's solution, 0.1% SDS and 100 µg / ml denatured salmon seed DNA and 5 ng / ml labeled probe. The blots were then washed for 10 minutes at room temperature in 2X SSC, ten minutes at 50 ° C for 1X SSC, followed by 10-15 minutes at 0.5X SSC.

·· ·· ·· · · · · ·

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Using a rat gene-derived probe, predominant mRNA species with a relative molecular weight of 2.4 kb were detected in several tissues. These tissues were kidney, liver, placenta'heart. The highest levels were detected in the kidneys. Large 4.5 and 7.5 kb mRNA species were detected in skeletal muscle and pancreas. It has been found that relatively high levels of 2.4 kb mRNA are expressed in human fetal tissue, the kidneys. Using a human probe (see below), only a 2.4 kb transcript was detected in the same tissues. Relatively high levels of the 2.4 kb transcript were also found in the lymph nodes, thymus, spleen and appendix. The size of the transcript, determined by both the rat and human osteosprotegerin gene, is mostly identical to the length of the rat pB1.1 FRI-1 insert, indicating that it is a full-length cDNA clone.

Example 3

Systemic delivery of OPG in transgenic mice

Rat OPG clone pB1. 1 was used as a template for PCR amplification of the coding region for subcloning into the ApoE liver specific expression vector (Simonet et al.,

J. Clin. Invest. 94, 1310-1319 (1994) and PCT application

No. US94 / 11675 and No. 08 / 221,767). The following 5 'and 3' oligonucleotide primers were used for PCR amplification:

5'-GACTAGTCCCACAATGAACAAGTGGCTGTG-3 '(SEQ ID NO: 9)

5 '-ATAAGAATGCGGCCGCTAAACTATGAAACAGCCCAGTGACCATTC-3' (SEQ ID NO: 10)

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4 · 4 · 4 4

4,444 · <44

4445 44 4 4

The PCR reaction mixture (Boehringer-Mannheim) was treated as follows: 1 minute at 94 ° C, 1 cycle;

seconds at 94 ° C, 30 seconds at 62 ° C and 1 minute at 74 ° C, -25 cycles. After amplification, samples were purified on Qiagen PCR columns and digested overnight with SpeI and NotI restriction enzymes. The digested products were extracted, precipitated, and subcloned into the ApoE promoter expression vector. Prior to microinjection of the resulting clone, HE-OPG, this clone was sequenced to ensure the absence of mutations.

The HE-OPG plasmid was purified by two cycles of CsCl density gradient centrifugation. Purified plasmid DNA was digested with XhoI and Ase I, and the 3.6 kb transgenic insert was purified by gel electrophoresis. The purified fragment was diluted to form a stock injection solution of 1 gg / ml in 5 mM Tris, pH 7.4, 0.2 mM EDTA. Single-cell embryos from BDF1 x BDF1-grown mice were injected essentially as described by Brinster et al. v Why. Nati. Acad. Sci. USA 82, 4338 (1985)) except that the injection needles were trimmed and siliconized prior to use. The embryos were grown overnight in a CO ₂ incubator and 15-20 double-cell embryos were transferred to the fallopian tubes of pseudogravid CD1 female mice.

After determination of pregnancy, 49 offspring were obtained from implantation of microinjected embryos. The offspring were screened by PCR amplification of the integrated transgene in genomic DNA samples. The targeted region for amplification was the 369 bp region of the human Apo E intron that is part of the expression vector. The oligomeric proteins used for PCR amplification were:

5'-GCC TCT AGA AAG AGC TGG GAC-3 '(SEQ ID NO: 11)

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5'-CGC CGT GTT CCA TTT ATG AGC-3 '(SEQ ID NO: 12)

The following conditions were used for PCR: two minutes at 94 ° C, 1 cycle; one minute at 94 ° C, 20 seconds at 63 ° C and 30 seconds at 72 ° C, 30 cycles. Of the original 49 offspring, 9 were identified as positive transgenic PCR.

Five transgenic 8-10 days subjects (2, 11, 16, 17 and 28) and five control subjects (1, 12, 15, 18 and 30) were sacrificed and autopsy and pathological analysis was performed. The liver was isolated from the remaining four transgenic subjects by partial hepatectomy. In partial hepatectomy, the mice were numb and the liver lobe was removed surgically. Total cellular RNA was isolated from the liver of all transgenic pups and five negative control pups as described by McDonald et al. in Meth. Enzymol. 152: 219 (1987). Northern blots analysis was performed on these samples to determine the level of transgenic expression. Approximately 10 gg of total RNA from the liver of each animal, which was dissolved by electrophoresis denaturing gels (Ogden et al., Meth. Enzymol 152, 61 (1987)), was subsequently transferred to a HYBOND-N nylon membrane (Amersham) and probed with ³² p dCTP-labeled pB1.1 insertion DNA. Hybridization was performed overnight at 42 ° C in 50% formamide, 5 x SSPE, 0.5% SDS, 5 x Denhardt's solution, 100 gg / ml denatured salmon seed DNA and 2-4 x 10 ⁶ cpm labeled probe / ml hybridization buffer. After hybridization was complete, the blots were washed twice for 5 minutes at room temperature in 2 x SSC followed by the same time in 0.1% SDS and then twice for 5-10 minutes at 55 ° C in 0.1 x SSC and 0.1% SDS. Transgene expression in the test and control pups was determined by the following autoradiography.

01-1718-97 Che 51 • · · · • · · · · • · · · • · · · • · · · · · • · · • · · · Data northernového blot suggest that 7 z transgenic individuals express e detectable level transgenic mRNA (animal no . 2, 11, 16, 17, 22 and 33 and 45). Negative control mice and one from transgenic mice

(# 28) did not express any transgene-related mRNA. Given that OPG is a secreted protein, overexpression of transgenic mRNA should represent the level of systemically delivered gene product. Mice 2, 17 and 22, which were positive for PCR and northern blots, expressed high levels of transgenic mRNA and could more biologically affect host cells and tissues.

Example 4

Biological activity of OPG

Five of the transgenic mice (animals 2, 11, 16, 17 and 28) and 5 control pups (animals 1, 12, 15, 18 and 30) were sacrificed for necropsy and pathological analysis. Prior to euthanasia, the identification numbers of all animals were verified, animals were weighed, numb and blood collected. Blood was stored both in the form of blood serum and in the form of complete blood for a complete chemical and hematological examination. Immediately after the anesthesia was terminated by lethal CO ₂ inhalation and before large units were separated, radiography was performed. Tissues were then removed and fixed in 10% buffered Zn-formalin for histological investigation. Collected tissues included liver, spleen, pancreas, stomach, duodenum, hip, intestine, spleen, reproductive organs, skin and mammary glands, bone, brain, heart, lungs, thymus, trachea, thyroid,

01-1718-97 Clavicle, rectum, adrenal gland, bladder and skeletal muscle. The liver, stomach, kidney, adrenal, spleen and thymus weights were determined prior to fixation. After fixation, the tissues were processed into paraffin blocks and 3 gm sections were obtained. Bone tissue was decalcified using a formic acid solution and all sections were stained with hematoxylin and eosin. Some tissues were also stained with Gomori reticulin and Masson trichrome. Enzymatic histochemistry was used to determine the expression of tartrate resistant acid phosphatase (TRAP), an enzyme highly expressed by multinucleated cells of monocyte-macrophage origin that resorb bone mass, called osteoclasts. To detect replicating cells, respectively. cells of monocyte-macrophage origin were also used for immunohistochemistry of the monocyte-macrophage surface of BrdU and F480 antigen. To detect F480 surface antigen expression, formalin-fixed, paraffin-encapsulated 4 μπι sections were deparaffinized and hydrated with deionized water. Sections were cooled with 3% hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh, PA) and incubated in rat monoclonal anti-mouse F480 antibody (Harlan, Indianopolis, IN). This antibody was detected using biotinylated-labeled rabbit anti-rat immunoglobulins, peroxidase-conjugated strepavidin (BioGenex San Ramon,

CA) with DAB as a chromagen (BioTek, Santa Barbara, CA) Sections were stained with a contrasting color, hematoxylin.

The separation of large units and examination of visceral tissues did not reveal any abnormalities in transgenic expressors or control pups. Organ weight analysis indicated that transgenic mice had approximately 38% splenic enlargement compared to controls. U • ·

01-1718-97 There was also a slight increase in platelet size and circulating unstained cells in transgenic expressors. In addition, transgenic receptors have been found to significantly reduce platelet levels. In addition, transgenic receptors have been shown to decrease serum uric acid levels, urine nitrogen levels, and alkaline phosphatase levels. Expression skeletons, including long bones (femur), vertebrae and flat bones (pelvic bones), have been found to exhibit increased radiohensity. The relative size of the femur in the expressors did not differ from that of the control mice.

Histological analysis of stained bone sections of OPG expressors showed severe osteopetrosis with the presence of cartilage residues of primary spongiosis apparent in the bone trabeculae, in the middle of the femur. A clearly defined bark was not identifiable in the femur sections. In normal animals, the central part of the bone is filled with bone marrow. Vertebra sections also show osteopetrotic changes, suggesting that OPG-induced skeletal changes were systemic. The remaining bone marrow showed mostly myeloid elements. Megakaryocytes were also present, Reticulin spots did not indicate reticulin deposition. Immunohistochemistry for the F4 80 cell surface antigen, expressed by monocyte-macrophage derivative cells in the mouse, indicated the presence of F480 positive cells in the medulla. Flattened F480 positive cells can be seen directly next to the beam-like bone surfaces.

Thighbone mesenchymal cells were flattened and appeared to be inactive. The distribution of H&E and TRAP spots revealed that in OPG expressors osteoclasts are rarely found on thigh bone surfaces.

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Osteoclasts and / or chondroclasts were found more in the area of cartilage-resorbing growth discs, but their numbers may be lower compared to controls. Osteoclasts were also present on the cortical surface of metaphyses, where construction activity is generally significant. The main difference between the expressors and the controls represented a substantial reduction in the trabecular osteoclasts in both the vertebrae and the femur. The extent of bone accumulation was directly proportional to the level of OPG transgenic mRNA detected by northern transfer of total liver RNA.

An increased amount of red marrow was found in the spleen of OPG expressors. This expansion is caused by increased hematopoiesis. All hematopoietic lines are represented. F480 positive cells were present in the red pulp of both control and OPG expressors. Two of the expressors (2 and 17) had extramedullary hematopoietic lesions in the liver, possibly due to osteopetrotic marrow.

No abnormalities were observed with the thymus, lymph nodes, gastrointestinal tract, pancreatohepatobiliary tract, respiratory tract, reproductive system, genitourinary system, skin, nervous system, heart and aorta, breast, skeletal muscle and fat.

Example 5

Isolation of mice and human OPG cDNA

A cDNA clone corresponding to the 5 'end of the mouse OPG mRNA was isolated by PCR amplification from mouse kidney cDNA library (Clonetech). The following oligonucleotides were derived from the rat OPG cDNA sequence:

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5'-ATCAAAGGCAGGGCATACTTCCTG-3 '5'-GTTGCACTCCTGTTTCACGGTCTG-3'

5'-CAAGACACCTTGAAGGGCCTGATG-3 '' -TAACTTTTACAGAAGAGCATCAGC-3 '

• ··· · • (SEQ ID NO: 13) (SEQ ID NO: 14) (seq ID NO: 15) (SEQ ID NO: 16)

5'-AGCGCGGCCGCATGAACAAGTGGCTGTGCTGCG-3 '(SEQ ID NO: 17)

5'-AGCTCTAGAGAAACAGCCCAGTGACCATTCC-3 '(SEQ ID NO: 18)

The partial and full-length cDNA products obtained by this procedure were sequenced. The full length product was digested with Not I and Xba I and subsequently cloned into the pRcCMV plasmid vector (Invitrogen). The resulting plasmid was designated as pRcCMV-Mu-OPG. The nucleotide sequence of the cloned product was compared to the rat OPG cDNA sequence. Within the 1300 bp region overlapping the OPG LORF, the rat and mouse DNA sequences were approximately 88% identical. The mouse cDNA sequence contained 401 and LORF, which is comparable to the rat OPG protein sequence, and was shown to be approximately 94% identical without gaps. This indicates that the isolated murine cDNA sequence encodes the mouse OPG protein and that the sequence and structure is largely conserved throughout evolution. The mouse OPG protein sequence contains at its N-terminus an identical probable signal peptide and all four potential N-linked glycosylation sites are retained. ------- - Partial human OPG cDNA was cloned from the human kidney cDNA library using the following rat-specific oligonucleotides:

5'-GTG AAG CTG TGG AAG AAC CTG ATG-3 '(SEQ ID NO: 19)

5'-ATC AAA GGC AGG GCA TAC TTC CTG-3 '(SEQ ID NO: 20)

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This PCR product was sequenced and used to construct primers to amplify the 3 'end of the human cDNA using the human OPG genomic clone in the lambda as a matrix: ... - - -

5'-TCCGTAAGAAACAGCCCAGTGACC-3 '(SEQ ID NO: 29)

5'-CAGATCCTGAAGCTGCTCAGTTTG-3 '(SEQ ID NO: 21)

The amplified PCR product was sequenced and used together with the 5 'end sequence to design 5' and 3 'human-specific primers useful in amplifying the entire human OPG cDNA coding sequences:

5'-AGCGCGGCCGCGGGGACCACAATGAACAAGTTG-3 '(SEQ ID NO: 22)

5'-AGCTCTAGAATTGTGAGGAAACAGCTCAATGGC-3 '(SEQ ID NO: 23)

The full-length human PCR product was sequenced, then cloned directly into plasmid vector pRcCMV (Invitrogen) using Not I and Xba I. The resulting plasmid was designated as pRcCMV-human OPG. The nucleotide sequence of the cloned product was compared to the rat and mouse OPG cDNA sequences. In the 1300 bp region overlapping the OPG LORF, rat and mouse DNA sequences were approximately 78-88% identical to human OPG cDNA. The human OPG cDNA sequence also contained 401 and LORF, which was comparable to the rat and mouse protein sequences. The predicted human OPG protein was approximately 85% and 95%, respectively. approximately 90%, identical to rat and mouse protein. Sequence alignment of rat, mouse and human protein revealed no significant changes during development. It is believed that the human protein has an N-terminal signal peptide and 5 potential N-linked glycosylation sites, of which 4 remained between the rat and mouse OPG protein.

··

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The DNA and predicted amino acid sequence of mouse OPG are shown in Figures 9A and 9B (SEQ ID NO: 122). The DNA and predicted amino acid sequence of human OPG are shown in Figures 9C and 9D (SEQ ID NO: 124). A comparison of the rat, mouse and human OPG amino acid sequences is shown in Figures 9E and 9F.

Isolation of other human OPG cDNA clones revealed the presence of a base C to G replacement at position 103 of the DNA sequence shown in Figure 9C. This nucleotide change results in lysine substitution by asparagine at position 3 of the amino acid sequence shown in Figure 9C. The rest of the sequence in the clones containing this change was identical to the corresponding regions shown in Figures 9C and 9D.

Example 6

OPG modeling of three-dimensional structure

The N-terminal portion of OPG has homology to the extracellular portion of all known members of the TNFR superfamily (Figure IC). The most prominent motif in this region of the TNFR-derived genes is an approximately 40 amino acid cysteine-rich repeat sequence that is packaged into distinct structures (Banner et al., C e 11-7.3.431-445 ^ (1993)). This moiety is seen and displayed in four (range 3 to 6) tandem repeats (see Figure 1C) and is known to be part of a ligand bond (Beutler and van Huffel Science 264, 667-663 (1994) )). Typically, each repeat contains six spaced cysteine residues that are involved in the formation of three intradomain disulfide bonds, designated SS1, SS2 and SS3 (Banner et al., Ibid.). At some receptors, •

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for example TNFR2, CD30 and CD40, some of the repeat domains contain only two intra-chain disulfide bonds (SS1 and SS3).

The human OPG protein sequence was aligned with the profile of the TNFR1 extracellular domain using the methods described by Luethy et al., Ibid., And the results were plotted using PrettyPlot from the Wisconsin Package, Version 8.1 (Genetics Computer Group, Madison, WI) (Figure 10). ). This sequence alignment was then used to construct a three-dimensional (3-D) model of the human OPG N-terminal domain. For construction, the known 3-D structure of the p55 TNFR1 extracellular domain (Banner et al., Ibid.) Was used as a template. Atomic coordinates of the peptide backbone and the side chains of identical residues were copied from the crystalline structural coordinates of TNFR1. Thereafter, the remaining advertising coordinates and various side chains were generated using the LOOK program (Molecular Applications Group, Palo Alto, CA). The 3-D model was then purified by minimizing its conformational energy using LOOK.

It is expected that OPG will bind to a ligand analogously to other members of the TNFR family. For the purpose of modeling the interaction of OPG with its ligand, it is stimulated

The 3-D model ..... "OPG ligand has a crystalline structure

TNF-β. This figure was graphically represented (see Figure 11) by Molscript (Kraulis, J. Appl. Cryst. 24, 946-950, 1991). A model for the OPG complex of OPG / ligand with 3 ββ and 3 OPG molecules was constructed in which the relative positions of OPG were identical to TNFR1 in the crystalline structure. This model was then used to identify OPG residues that could interact with their ligand. For the survey he chose

01-1718-97 The following approach. The area of all solvent-accessible residues and one OPG model was calculated. Residues that have different accessibility in the complex than the monomer are likely to react with the ligand. The human and murine OPG amino acid sequences were rearranged using this information into the most prominent sequences, each containing cysteine rich domains 1-4 (Figures 12A and 12B). Each domain had unique structural properties that could be assumed.

Domain 1

It contains 4 cysteine residues contained in SS2 (C41 to C54) and SS3 (C44 to C62) disulfide bonds). Although no SS1 bond is apparent from the disulfide bridges, the tyrosine retained at position 28 is homologous to Y20 in TNFR1, which is known to be involved in the interaction with H66 and thus aid in domain formation. OPG has homologous histidine at position 75, suggesting that OPG Y28 and H75 accumulate together in the native protein just as homologous residues in TNFR1 do. Thus, both of these residues may in fact be important for biological activity, and N-terminal OPG treatments up to Y28 and beyond Y28 may alter activity. Based on the study of the three-dimensional model described above, it is expected that residues E34 and K43 interact with the binding ligand.

Domain 2

It contains six cysteine residues and can be assumed to contain SS1 (C65 to C80), SS2 (C83 to C80).

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C98) and SS3 (C87 to C105) disulfide bonds. This region of OPG also contains a region stretching from P66-Q91 which is aligned with the portion of TNFR1 domain 2 which forms close contacts with TNF.beta (see above) and may interact's ^OPG ligand. Based on the structural data obtained, it is expected that the following residues P66, H68, Y69, Y70, T71, D72, S73, H75, T76, S77, D78, E79, L81, Y82, P85, V86, K88 interact with the binding ligand. , E89, L90, and Q91.

Domain 3

It contains 4 cysteine residues that are part of SS1 (C107 to C118) and SS3 (C124 to C142) disulfide bonds but not SS2 bonds. Based on the obtained structural data, it can be assumed that residues E115, L118 and K119 interact with the OPG ligand.

Domain 4

It contains 4 cysteine residues that are part of SS1 (C145 to C160) and SS3 (C166 to C185) disulfide bonds but not SS2 bonds. Based on the obtained structural data, it is expected that residues E153 and S155 interact with the OPG ligand.

Thus, the putative OPG structural model has identified a number of highly conserved residues that are likely to be important for its biological activity.

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Example 7

Product of recombinant secreted OPG protein in mammalian cells

To determine whether OPG is a truly secreted protein, murine OPG cDNA was fused to a human IgG1 Fc labeling domain (Capon et al., Nature 337, 525-531 (1998)) and expressed in human 293 fibroblasts. Fc fusions were performed using the pFc-A3 vector. The vector pFc-A3 contains a region encoding the Fc region of the human immunoglobulin IgG-γΙ heavy chain (Ellison et al., Ibid.) From the first amino acid of the hinge domain (Glu-99) to the carboxyl terminus and flanked by a 5'-NotI fusion site and 3'- SalI and Xbal sites. The plasmid was constructed by PCR amplification of the human spleen cDNA library (Clontech). PCR reactions were performed in a final volume of 100 μΐ and utilized 2 units of Vent DNA polymerase (New England Biolabs) in 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 μΜ (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ ,

0.1% Triton X-100 with 400 μΜ single dNTP and 1 ng cDNA library to be amplified together with 1 μΜ of each primer. Reactions were initiated by a two minute denaturation at 95 ° C, followed by 30 cycles, including 30 seconds at 95 ° C, thirty seconds at 55 ° C, and two minutes at 73 ° C. 5 'primer

5 'ATAGCGGCCGCTGAGCCCAAATCTTGTGACAAAACTCAC 3' (SEQ ID NO: 24) incorporated the NotI site immediately after 5 'to the first residue (Glu-99) of the IgG-γ1 hinge domain. 3 'primer

5 '-TCTAGAGTCGACTTATCATTTACCCGGAGACAGGGAGAGGCTCTT-3' (SEQ ID NO: 25)

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built Sall and Xbal sites. The 717-bp PCR product was digested with NotI and SalI, isolated by electrophoresis through 1% agarose (FMC Corp.), purified by Geneclean (BIO 101 Inc) and cloned into NotI, SalI-digested pBluescript II KS vector (Stratagene). The insert in the resulting plasmid, pFcA3, was sequenced to confirm the accuracy of the PCR reaction.

The cloned mouse cDNA in the plasmid pRcCMV-MuOPG was amplified using the following two sets of primer pairs:

Pár 1

5'-CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3 '(SEQ ID NO: 26)

5'-CCTCTGCGGCCGCTAAGCAGCTTATTTTCACGGATTGAACCTG-3 '(SEQ ID NO: 27)

Couple 2

5'-CCTCTGAGCTCAAGCTTCCGAGGACCACAATGAACAAG-3 '(SEQ ID NO: 28)

5'-CCTCTGCGGCCGCTGTTGCATTTCCTTTCTG-3 '(SEQ ID NO: 30)

The first pair amplified the entire OPG LORF and generated a NotI restriction site that was compatible with the Not I site in the Fc fusion vector pFcA3. The pFcA3 vector was prepared by creating a Not I restriction site 5 ^of ? On the aspartic acid residue 216 of the human IgG1 Fc cDNA. This construct introduces a linker that encodes two irrelevant amino acids that overlap the link between the OPG protein and the IgG Fc region. This product, when bound to the Fc portion, could directly encode all 401 OPG residues and subsequently all 227 amino acid residues of the human IgG1 Fc region (Fl.Fc). The second primer pair amplifies the DNA sequences,

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encoding the first 180 amino acid residues of OPG that include their likely ligand binding domain. As mentioned above, the 3 'primer forms an artificial Not I restriction site that binds the C-terminal modified OPG LORF at position threonine 180 directly to the IgG1 Fc domain (CT.fc).

The linking of the amino acid sequence binding OPG residue 401 and aseptic acid residue 221 of the human Fc region can be modified as follows: DNA encoding residues 216-220 of the human Fc region can be deleted as described above, or the cysteine residue corresponding to the C220 human Fc region can be mutated to either serine or alanine. The OPG-Fc fusion protein encoded by these modified vectors can be transfected into human 293 cells or CHO cells and the recombinant OPG-Fc fusion protein purified as follows.

Both products were directly cloned into plasmid vector pCEP4 (Invitrogen). The pCEP4 vector contains a replication source, which is Epstein-Barr virus, and is capable of episomal replication in 293-EBNA-1 cells. Parental pCEP4, pCEP4-F1.Fc and pCEP4-CT.Fc vectors were lipofected into 293-EBNA-1 cells using the methods recommended by the manufacturer. The transfected cells were subsequently picked in 100 gg / ml hygromycin to select for vector expression and the resulting drug-resistant mass cultures grew until they formed whole. The cells were then grown for 72 hours in serum free medium, after which the conditioned medium was removed and analyzed by SDS-PAGE. The silver staining of the polyacrylamide gel detected the major proteins of the conditioned medium produced for the drug by resistant 293 cultures. In pCEP4-Fl.Fc and pCEP4-CT.Fc conditioned media, they were heavily secreted.

01-1718-97 Unique belts of predicted sizes (see figures 13B and 13C). The full-length Fc fusion protein has accumulated to a high concentration, indicating that it may be stable. Both Fc fusion proteins were detected by anti-human IgG1 βc antibodies (Pierce) on western blots, suggesting that they formed recombinant OPG products.

The full-length OPG-Fc fusion protein was purified by Protein-A column chromatography using the procedures recommended by the manufacturer. The protein was subjected to N-terminal sequence analysis using the automated Edmann degradation essentially described by Matsudair et al. (J. Biol. Chem. 26, 10-35 (1987)). After 19 cycles, the following amino acid sequence was read:

NH2-E TLPPKYLHYDPETGHQL L-O 2 H (SEQ ID NO: 31)

This sequence is identical to the putative murine OPG amino acid sequence starting with amino acid residue 22, suggesting that in mammals the major site of natural cleavage is between amino acid residues Q21-E22 and not between Y31-D32 as originally predicted. Expression experiments performed in 293-EBNA cells using pCEP4-F1.Fc and pCEP4-CT.Fc show that OPG is secreted by protein and can act systemically. in terms of binding to its ligand.

Procedures similar to those used to construct and express muOPG [22-180] -Fc and muOPG [22-401] -Fc fusions were also used for other mouse and human OPG-Fc fusion proteins.

A mouse OPG cDNA encoding amino acids 1-185 bound to the Fc region of human IgG1 [muOPG Ct (185) .Fc] was constructed.

01-1718-97 In the following way. Mouse OPG cDNA from pRcCMV Mu osteoprotegerin (described in Example 5) was amplified as described above using the following primer pair in the polymerase chain reaction:

1333-82:

5'-TCC CTT GCC CTG ACC ACT CTT-3 '(SEQ ID NO: 32) 1333-80:

5'-CCT CTG CGG CCG ACA CGT TGT CAT GTG TTG 0-3 '(SEQ ID NO: 33)

This primer pair amplifies the mouse OPG cDNA region encoding amino acid residues 63-185 (corresponding to bp 278-645) of the OPG reading frame, as shown in Figure 9A. The 3 'primer contains a Not I restriction site that is compatible with the flanked Not I Fc site of the pFcA3 fusion vector. The product also overlaps a unique EcoRI restriction site, located at bp 436. The amplified PCR product was purified, digested with NotI and EcoRI, and the resulting EcoRI-NotI restriction fragment purified. The vector pCEPP4, which had a mouse 1-401 OPG-Fc fusion insert, was digested with EcoRI and NotI, purified and sequenced with the above generated PCR product. The resulting pCEP4-based expression vector directly encodes OPG residues 1-185 followed by 227 amino acid residues of the human IgG1 Fc region. Mouse OPG 1-185. The Fc fusion vector was transfected into 293 cells, the drug was selected and conditioned media prepared as described above. The resulting secreted mouse OPG 1-185.Fc fusion product was purified by Protein-A column chromatography (Pierce) using procedures recommended by the manufacturers.

01-1718-97 Če

Mouse OPG DNA encoding amino acid residues 1-194 cloned into the Fc region of human IgG1 (muOPG Ct (194) .Fc) was constructed as follows. Mouse OPG cDNA from plasmid pRcCMV Mu-osteoprotegerin was amplified using the following primer pairs:

1333-82:

5'-TCC GTC CTG ACC ACT CTT-3 '(SEQ ID NO: 34)

1333-81:

5'-CCT CTG CGG CCG CCT TTT GCG TGG CTT CTC TGT T-3 '(SEQ ID NO: 35)

This primer pair amplifies the mouse OPG cDNA region encoding amino acid residues 70-194 (corresponding to bp 298-672) of the OPG reading frame. The 3 'primer contains a Not I restriction site that is compatible with the flanked Not I Fc site of the pFcA3 fusion vector. The product also extends beyond the unique EcoRI restriction site located at bp 436. The amplified PCR product was cloned into the mouse OPG [1-401] Fc fusion vector described above. The resulting pCEP4-based expression vector directly encodes OPG residues 1 to 194 followed by 227 amino acid residues of the human IgG1 Fc region. The mouse OPG 1-194.Fc fusion vector was transfected into 293 cells, the drug was selected and conditioned medium was prepared as described above. Resulted secretary. the fusion product was purified by Protein-A column

chromatography manufacturers. (Pierce) using recommended procedures Humane OPG DNA, coding amino acids 1-401, tilted to Fc segment humánního IgG1 was constructed

as follows. Human OPG DNA in plasmid pRcCMV-hu

01-1718-97 osteoprotegerin (described in Example 5) was amplified using the following oligonucleotide primers:

1254-90:

5'-CCT CTG AGC (SEQ ID NO: 37) TCA AGC TTG GTT TCC GGG GAC CAC AAT G-3 ' 1254-95: 5'-CCT CTG CGG CCG CTA AGC AGC TTA TTT TTA CTG AAT GG-3 ' (SEQ ID NO: 38) Resultant The PCR product encodes full-length human OPG

protein and forms a NoT I restriction site that is compatible with the flanked Not I Fc site of the FcA3 fusion vector. The PCR product was directly cloned into the above described plasmid vector pCEP4. The resulting expression vector directly encodes human OPG residues 1-401, followed by 227 amino acid residues of the human IgG1 Fc region. Conditioned medium was prepared from transfected and drug-selected cells and the huOPG Fl.Fc fusion product was purified by Protein-A column chromatography (Pierce) using procedures recommended by the manufacturers.

Human OPG DNA encoding amino acid residues 1 to 201, encoded into the Fc region of human IgG1 [huOPG Ct (201) .Fc], was constructed as follows. Human OPG cDNA cloned from plasmid pRcCMV-hu osteoprotegerin was amplified by PCR using the following oligonucleotide primer pair:

1254-90:

5'-CCT CTG AGC TCA AGC TTG GTT TCC GGG GAC CAC AAT G-3 '(SEQ ID NO: 38)

01-1718-97 Če

1254-92:

5'-CCT CTG CGG CCG CCA GGG TAA CAT CTA TTC CAC-3 '(SEQ ID NO: 39)

This primer pair amplifies the human OPG cDNA region encoding amino acid residues 1-201 of the OPG reading frame, and creates a Not I restriction site at the 3 'end that is compatible with the flanked Not I Fc site of the FcA3 fusion vector. This product, when bound to the Fc region, directly encodes OPG residues 1-201 and subsequently all 221 amino acid residues of the human IgG1 Fc region. The PCR product was directionally cloned into the pCEP4 plasmid vector as described above. Conditioned medium and hu OPG Ct (201) were prepared from transfected and drug-selected cells. Fc fusion products were purified by Protein-A column chromatography (Pierce) using procedures recommended by the manufacturers.

The following procedures were used to construct and express non-fused mouse and human OPG.

A mammalian full-length mouse OPG expression plasmid (residues 1-401) was generated by PCR amplification of the mouse OPG cDNA insert from pRcCMV Mu-osteoprotegerin and subcloned into the pDSRα expression vector (DeClerck et al., J. Biol. Chem. 266, 3893 (1991)). ). The following oligonucleotide primers were used for this purpose:

1295-26:

5'-CCG AAG CTT CCA TCA AGA AGT GGC TGT GCT GC-3 '(SEQ ID NO: 40)

1295-27:

01-1718-97 Če

5'-CCT CTG TCG ATT ATA ATC AGC AGC TTA TTT TCA CGG ATT G-3 '(SEQ ID NO: 41)

The mouse OPG full-length reading frame was amplified by the PCR described above. The PCR product was purified and digested with restriction endonuclease Hind III and Xba I (Boehringer Mannheim, Indianapolis, IN) under the manufacturer's recommended conditions, and then ligated with Hind III and Xba I to digested pDSRα. Recombinant clones were detected by digestion with restriction endonuclease and subsequently sequenced to avoid mutations during PCR amplification steps.

Single expressions

The resulting plasmid, pDSRa-muOPG, was introduced into Chinese hamster ovary (CHO) cells by calcium mediated transfection (Wigler et al., Cell 11, 233 (1977)) colonies were selected based on the dihydrofolate reductase (DHFR) gene in the plasmid vector and several clones is isolated. Mouse OPG recombinant protein expression was monitored by Western blotting of conditioned CHO cell media. Highly expressing cells were selected and OPG expression was further amplified with methotrexate as described by DeClerck et al., Ibid. Conditioned medium from CHO cell lines was further purified for recombinant secreted mouse OPG protein.

A plasmid for mammalian expression of full-length human OPG (amino acids 1-401) was generated by subcloning the cDNA insert in pRcCMV-hu osteoprotegerin directly into the pDSRα vector (DeClerck et al., Ibid.). Plasmid pRcCMV-OPG was completely digested with Not I, with Klenow blunted ends and then completely digested with Xba I. The DNA vector was

01-1718-97 Hind digested with Klenow, then digested with Xba I and ligated to OPG insert. The recombinant plasmids were then sequenced to confirm the correct orientation of the human OPG cDNA.

The resulting plasmid pDSRa-huOPG was introduced into hamster ovary (CHO) cells, as mentioned above. Individual colonies were selected based on the expression of the dihydrofolate reductase (DHFR) gene in the plasmid vector, and several clones were isolated. Human OPG recombinant protein expression was monitored by western blotting of conditioned CHO cell media. Highly expressing clones were selected and OPG expression was further amplified with methotrexate. For protein purification, a conditioned medium was prepared from CHO cell lines expressing human OPG.

Mouse OPG expression vectors encoding residues 1 to 185 were constructed as follows. Mouse OPG cDNA from pRcCMV-Mu OPG was amplified using the following oligonucleotide primers:

1333-82:

5'-TCC GTC CTG ACC ACT CTT-3 '(SEQ ID NO: 42)

1356-12:

5'-CCT CTG TCG ACT TAAAAC ACG TTG TCA TGT GTT GC-3 '(SEQ ID NO: 43)

This primer pair amplifies the mouse OPG cDNA region encoding amino acids 63 to 185 of the OPG reading frame (bp 278645) and contains an artificial tail codon directly after the cysteine codon (C 185) followed by an artificial Sal I restriction

01-1718-97 Če

endonuclease site. The putative product contains an internal Eco RI restriction site useful for subcloning into an existing vector. After PCR amplification, the resulting purified product was digested with Eco RI and Sal I restriction endonuclease and the large fragment was gel purified. The purified product is then subcloned into the large Eco RI and Sal I restriction fragment of the pBluescript-muOPG Fl.Fc. digest described above. The resulting plasmid was digested with Hind III and Xho I, and the small fragment was gel purified. This fragment, which contains an open reading frame encoding residues 1 to 185, was subsequently subcloned into the Hind III and Xho I digests of the pCEP4 expression vector. The resulting vector, pmuOPG [1 to 185], encodes a modified OPG polypeptide that terminates at the cysteine residue located at position 185. As mentioned above, conditioned medium was prepared from transfected and drug-selected cells.

1333-82:

5'-TCC GTC CTG ACC ACT CTT-3 '(SEQ ID NO: 44)

1356-13:

5'-CCT CTG TCG TCT TTT TGC GTG GCT TCT CTG TT-3 '(SEQ ID NO: 45)

This primer pair amplifies the mouse OPG cDNA region encoding amino acids 70-194 of the OPG reading frame (bp 298-672) and contains an artificial tail codon directly after the lysine codon (K194) followed by an artificial Sal I restriction endonuclease site. The envisaged product contains an Eco RI restriction site useful for subcloning into an existing vector. After PCR amplification, the resulting purified product was cleaved.

01-1718-97 with Eco RI and Sal I restriction endonuclease and the large fragment was gel purified. The purified product is then subcloned into the large Eco RI and Sal I restriction fragment of the pBluescript-muOPG Fl.Fc. digest described above. The resulting plasmid was digested with Hind III and Xho I, and the small fragment was gel purified. This fragment, which contains an open reading frame encoding residues 1 to 185, was subsequently subcloned into the Hind III and Xho I digests of the pCEP4 expression vector. The resulting vector, pmuOPG [1 to 185], encodes a modified OPG polypeptide that terminates at the cysteine residue located at position 185. As mentioned above, conditioned medium was prepared from transfected and drug-selected cells.

Several mutations were generated at the 5 'end of the huOPG [22-401] -Fc gene that introduces either amino acid substitutions or OPG deletions between residues 22-32. All mutations were generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, San Diego) , CA) using the manufacturer's recommended conditions. Briefly, the reaction mixture containing huOPG [22-401] -Fc, plasmid DNA matrix and mutagenic primers was treated with Pfu polymerase in the presence of deoxynucleotides and then amplified as described above in a thermocycler. An aliquot of the reaction mixture was then heat-transfected into competent E. coli XL1-blue and mutation verification was performed. Plasmid DNA from the transformants was subsequently sequenced for various mutations.

The following primer pairs were used to delete residues 22-26 of the human OPG gene that resulted in the huOPG [27-401] -Fc fusion protein:

··

01-1718-97 Če

1436-11:

5'-TGG ACC ACC CAG AAG TAC TAT CAT TAT GAC-3 '(SEQ ID NO: 140)

1436-12:

5'-GTC ATA ATG AAG GTA CTT CTG GGT GGT CCA-3 '(SEQ ID NO: 141)

The following primer pairs were used for the deletion of residues 22-28 of the human OPG gene, which resulted in huOPG [29-401] -Fc fusion protein:

1436-17:

5'-GGA CCA CCC AGC TTC ATT ATG ACG AAG AAA-3 '(SEQ ID NO: 142)

1436-18:

5'-GTT TCT TCG TCA TAA TGA AGC

The following primer pairs were used to delete residues 22-31 of the human OPG gene that resulted in the huOPG [32-401] -Fc fusion protein:

1436-27:

5'-GTG GAC CAC CCA GGA CGA AGA AAC TC-3 '(SEQ ID NO: 144) _

1436-28:

5 '-GAG AGG TTT CTT CGT CCT GGG TGG GGT TCC-3' (SEQ ID NO: 145)

The following primer pairs were used to change the codon for tyrosine residue 28 to phenylalanine of human OPG

01-1718-97 • g mělo g g,,,,,,,,,,,,,,,, resulting in the production of huOPG [22-401] -Fc Y28F fusion protein:

1436-27:

5'-CGT TTC CTC CAA AGT TCC ATT ATG AC-3 '(SEQ ID NO: 146)

1436-30:

5 '-GTC ATA ATG AAG GAA CTT TGG AGG AAA CG-3' (SEQ ID NO: 147)

The following primer pairs were used to change the codon for tyrosine residue 26 to the alanine of the human OPG gene, resulting in the production of huOPG [22-401] -Fc P26A fusion protein:

1429-83:

5'-GGA ACC GTT TCC TGA AAA GTA CCT TCA TG-3 (SEQ ID NO: 148)

1429-84:

5'-CAT AAT GAA ACG TTT CAG GAA ACG TTT CC -3 '(SEQ ID NO: 149)

Each resulting muOPG [22-401] -Fc plasmid containing the respective mutation was subsequently transfected into human 293 cells and the mutant OPG-Fc fusion protein was purified from the above conditioned media. The biological activity of each protein was tested in vitro by the osteoclast formation assay described in Example 11.

01-1718-97 Če

Example 8

OPG expression in E. coli

A. Bacterial pAMG21 expression vectors

The expression plasmid pAMG21 may be derived from the Amgen expression vector system pCFM1656 (ATCC # 69576), which in turn may be derived from the Amgen expression vector system described in US Patent 4,710,473. Plasmid pCFM1656 can be derived from the described pCFM36 plasmid (US Patent 4,710,473) by: (a) destroying two endogenous NdeI restriction sites by end-filling with T4 polymerase enzyme and subsequently adjusting the end of the ligation; (b) replication of the DNA sequence between the unique AatII and ClaI restriction sites containing the synthetic P _L promoter with a similar fragment obtained from pCFM636 (US Patent 4,710,473) containing the PL promoter

AatlI

5 'CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT3 · TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA-AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-TTTTTTGTATGTCTATTGGTAGACGCCAGTAŤTTAATAGAGACGGCCACAACTGTATTT TACCACTGGCGGTGATACTGAGCACAT-3' (SEQ ID NO: 53)

-ATGGTGACCGCCACTATGACTCGTGTAGC5 '(SEQ ID NO: 54)

Clal followed by (c) substituting a small DNA sequence between the unique Clal and KpnI restriction sites by the following oligonucleotides:

01-1718-97 Če

• 9 ·· ·· ·· • * • • · • • · • · • • · · · • • Λ • • • • • • 9 ·· • ·

5 '(SEQ

3 '(SEQ

CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC3 'ID NO: 48)

TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5 '

NO ID: 49)

Clal

- KpnI

The expression plasmid can then be derived from pCFM1656 by preparing a series of site-directed basic PCR changes, overlapping oligomutagenesis and DNA sequence substitutions. Beginning with the BglII site (plasmid bp # 180) immediately 5 'to the PcopB plasmid replication promoter towards the plasmid replication genes, the following base pair changes occurred:

pAMG21 bp # bp in pCFM1656 bp in pAMG21 changed to # 204 THE C / G # 428 A / T G / C # 509 G / C A / T # 617 - - two ads G / C bp # 679 G / C THE # 980 THE C / G # 994 G / C A / T # 1004 A / T C / G # 1007 C / G THE # 1028 A / T THE # 1047 “·” ^{! == i} C / G ^s ' T / A = ⁵ - # 1178 G / C THE # 1466 G / C THE # 2028 G / C bp deletion # 2187 C / G THE # 2480 A / T THE

* ·· ·· ·· ·· · · · · · · · · · · ·

01-1718-97 Če 77 « • ···· bp v • · · · · · · · · · · · · · · · · pAMG21 changed to pAMG21 bp # bp v PCFM1656 # 2499-2502 AGTG. TCAC - -........ - GTCA CAGT • # # # 2642 3435 3446 3643 TCCGAGC AGGCTCG G / C G / C A / T 7 bp A / T A / T THE deletions DNA sequence among unique ones AatlI (Position # 4364 in

pCFM1656) and SacII (position # 4585 in pCFM1656) restriction sites is substituted with the following DNA sequence:

[AatII Cohesive End] ₅ , GCGTAACGTATGCATGGTCTCC (position # 4358 in pAMG21) 5 'TGCACGCATTGCATACGTACCAGAGG—

-CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACT-GGTACGCTCTCATCCCTTGACGGTCCGTAGTTTATTTTGCTTTCCGAGTCAGCTTTCTGA-GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGC-CCCGGAAAGCAAAATAGACAACAAACAGCCACTTGCGAGAGGACTCATCCTGTTTAGGCG-CGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGC-GCCCTCGCCTAAACTTGCAACGCTTCGTTGCCGGGCCTCCCACCGCCCGTCCTGCGGGCG-CATAAaCTGCCAGGCATCáAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT-GTATTTGACGGTCCGTAGTTTAATTCGTCTTCCGGTAGGACTGCCTACCGGAAAAACGCAAatlI

-TTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGGACGTCGTACTTAAC-AAGATGTTTGAGAAAACAAATAAAAAGATTTATGTAAGTTTATACCTGCAGCATGAATTG-TTTTAAAGTATGGGCAATCAATTGCTCCTGTTAAAATTGCTTTAGAAATACTTTGGCAGC-AAAATTTCATACCCGTTAGTTAACGAGGACAATTTTAACGAAATCTTTATGAAACCGTCG-GGTTTGTTGTATTGAGTTTCATTTGCGCATTGGTTAAATGGAAAGTGACCGTGCGCTTAC-CCAAACAACATAACTCAAAGTAAACGCGTAACCAATTTACCTTTCACTGGCACGCGAATG • ·

-1718-97 Če

-TACAGCCTAATATTTTTGAAATATCCCAAGAGCTTTTTCCTTCGCATGCCCACGCTAAAC-ATGTCGGATTATAAAAACTTTATAGGGTTCTCGAAAAAGGAAGCGTACGGGTGCGATTTG-ATTCTTTTTCTCTTTTGGTTAAATCGTTGTTTGATTTATTATTTGCTATATTTATTTTTCTAAGAAAAAGAGAAAACCAATTTAGCAACAAACTAAATAATAAACGATATAAATAAAAAG-GATAATTATCAACTAGAGAAGGAACAATTAATGGTATGTTCATACACGCATGTAAAAATA CTATTAATAGTTGATCTCTTCCTTGTTAATTACCATACAAGTATGTGCGTACATTTTTAT • • • AACTATCTATATAGTTGTCTTTCTCTGAATGTGCAAAACTAAGCATTCCGAAGCCATTAT-TTGATAGATATATCAACAGAAAGAGACTTACACGTTTTGATTCGTAAGGCTTCGGTAATATAGCAGTATGAATAGGGAAACTAAACCCAGTGATAAGACCTGATGATTTCGCTTCTTTAAatcgtcatacttatccctttgatttgggtcactattctggactactaaagcgaagaaattTTACATTTGGAGATTTTTTATTTACAGCATTGTTTTCAAATATATTCCAATTAATCGGTGAATGTAAACCTCTAAAAAATAAATGTCGTAACAAAAGTTTATATAAGGTTAATTAGCCAC AATGATTGGAGTTAGAATAATCTACTATAGGATCATATTTTATTAAATTAGCGTCATCATTTACTAACCTCAATCTTATTAGATGATATCCTAGTATAAAATAATTTAATCGCAGTAGTAAATATTGCCTCCATTTTTTAGGGTAATTATCCAGAATTGAAATATCAGATTTAACCATAGTTATAACGGAGGTAAAAAATCCCATTAATAGGTCTTAACTTTATAGTCTAAATTGGTATCAATGAGGATAAATGATCGCGAGTAAA • TAATATTCACAATGTACCATTTTAGTCATATCAG TTACTCCTATTTACTAGCGCTCATTTATTATAAGTGTTACATGGTAAAATCAGTATAGTCATAAGCATTGATTAATATCATTATTGCTTCTACAGGCTTTAATTTTATTAATTATTCTGTTATTCGTAACTAATTATAGTAATAACGAAGATGTCCGAAATTAAAATAATTAATAAGACAAAGTGTCGTCGGCATTTATGTCTTTCATACCCATCTCTTTATCCTTACCTATTGTTTGTCTTCACAGCAGCCGTAAATACAGAAAGTATGGGTAGAGAAATAGGAATGGATAACAAACAGGCAAGTTTTGCGTGTTATATATCATTAAAACGGTAATAGATTGACATTTGATTCTAATAACGTTCAAAACGCACAATATATAGTAATTTTGCCATTATCTAACTGTAAACTAAGATTATTATTGGATTTTTGTCAGACTATTATATCGCTTGAAATACAATTGTTTAACATAAGTACCTGTAACCTAAAAACAGTGTGATAATATAGCGAACTTTATGTTAACAAATTGTATTCATGGACTAGGATCGTACAGGTTTACGCAAGAAAATGGTTTGTTATAGTCGATTAATCGATTTGATTATCCTAGCATGTCCAAATGCGTTCTTTTACCAAACAATATCAGCTAATTAGCTAAACTAACTAGATTTGTTTTAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGATCTAAACAAAATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTSacII

Zhejiang GCTCACTAGTGTCGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAÁCGAGTGATCACAGCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTTGAAGAAGAAGAAGAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATACTTCTTCTTCTTCTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTATACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGTGATCGTATTGGGGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCC01-1718-97

-AACCGCTCTTCACGCTCTTCACGC 3 '-TTGGCGAGAAGTGCGAGAAGTG 5' [SacII Cohesive End] (SEQ ID NO: 50) (Position # 5904 in pAMG21) (SEQ ID NO: 46)

During ligation of the cohesive end of this substituted DNA sequence, the outer AatII and SacII sites are destroyed. There are unique AatIII and SacII sites in the substituted DNA.

pAMG22-His

The pAMG22-His expression plasmid can be derived from the Amgen expression vector pAMG22 by substituting a small DNA sequence between the unique NdeI (# 4795) and EcoRI (# 4818) restriction sites by the following oligonucleotide duplex:

Ndel -NtlSl. EcoRI

5 'TATGAAACATCATCACCATCACCATCATGCTAGCGTTAACGCGTTGG 3' (SEQ ID NO: 51) 'ACTTTGTAGTAGTGGTAGTGGTAGTACGATCGCAATTGCGCAACCTTAA 5' (SEQ ID NO: 52)

MetLysHisHisHisHisHisHisHisAlaSerValAsnAlaLeuGlu (SEQ ID NO: 168) pAMG22

The expression plasmid pAMG22 can be derived from the Amgen expression vector pCFM1656 (ATCC # 69576), which in turn can be derived from the Amgen expression vector system described in US Patent 4,710,473, granted

December 1, 1987. Plasmid pCFM1656 can be derived from

01-1718-97 of the above described plasmid pCFM836 (US Patent 4,710,473): (a) destroying two endogenous NdeI restriction sites by end-filling with T4 polymerase enzyme and then blunting the end of the ligation; (b) replication of the DNA sequence between the unique AatII and ClaI restriction sites containing a synthetic P _L promoter with a similar fragment obtained from pCFM636 (US Patent 4,710,473) that contains the PL promoter

Aatix

5 'CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT3' TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA-AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT TACCACTGGCGGTGATACTGAGCACAT-3 '(SEQ ID NO: 53) -ATGGTGACCGCCACTATGACTCGTGTAGC5' (SEQ ID NO: 54)

Clal followed by (c) substituting a small DNA sequence between the unique Clal and KpnI restriction sites by the following oligonucleotides:

5 'CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3' (SEQ ID NO: 55)

3 'TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5' (SEQ ID NO: 56) == ---- Clal KpnI

The expression plasmid pAMG22 can then be derived from pCFM1656 by preparing a series of site-directed basic PCR changes by overlapping oligomutagenesis and DNA sequence substitutions. Starting with the BglII site (plasmid

Bp # 180) immediately 5 'to the PcopB plasmid replication promoter towards the plasmid replication genes, with the following base pair changes:

pAMG22 bp # bp in pCFM1656 bp in pAMG22 changed to J # 204 THE C / G J # 428 A / T G / C K # 509 G / C A / T K # 617 - - two ads G / C bp K # 679 G / C THE J # 980 THE C / G J # 994 G / C A / T J # 1004 A / T C / G J # 1007 C / G THE J # 1028 A / T THE J # 1047 C / G THE J # 1178 G / C THE J # 1466 G / C THE K # 2028 G / C bp deletion J # 2187 C / G THE H # 2480 A / T THE ... acmc .... ctcs. . . . .. TCAC CAGT J # 2642 TCCGAGC AGGCTCG 7 bp deletion J # 3435 G / C A / T J # 3446 G / C A / T J # 3643 A / T THE

• ·

01-1718-97 Če

The DNA sequence between the unique AatII (position # 4364 in PCFM1656) and SacII (position # 4585 in pCFM1656) restriction sites is substituted with the following DNA sequence:

[AatII cohesive end] (position # 4358 in pAMG22) 'GCGTAACGTATGCATGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAA3' TGCACGCATTGCATACGTACCAGAGGGGTACGCTCTCATCCCTTGACGGTCCGTAGTT-ATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG-TATTTTGCTTTCCGAGTCAGCTTTCTGACCCGGAAAGCAAAATAGACAACAAACAGCCAC-AACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGG-TTGCGAGAGGACTCATCCTGTTTAGGCGGCCCTCGCCTAAACTTGCAACGCTTCGTTGCC-CCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAG-GGGCCTCCCACCGCCCGTCCTGCGGGCGGTATTTGACGGTCCGTAGTTTAATTCGTCTTC-GCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAAT-CGGTAGGACTGCCTACCGGAAAAACGCAAAGATGTTTGAGAAAACAAATAAAAAGATTTAAatlI

-ACATTCAAATATGGACGTCTCATAATTTTTAAAAAATTCATTTGACAAATGCTAAAATTC-TGTAAGTTTATACCTGCAGAGTATTAAAAATTTTTTAAGTAAACTGTTTACGATTTTAAG-TTGATTAATATTCTCAATTGTGAGCGCTCACAATTTATCGATTTGATTCTAGATTTGTTT-AACTAATTATAAGAGTTAACACTCGCGAGTGTTAAATAGCTAAACTAAGATCTAAACTCA-TAACTAATTAAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGAGCTCACTAGTGT-ATTGATTAATTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGCTCGAGTGATCACASacII

-CGACCTGCAGGGTACCATGGAAGCTTACTCGAGGATCCGCGGAAAGAAGAAGAAGAAGAA-GCTGGACGTCCCATGGTACCTTCGAATGAGCTCCTAGGCGCCTTTCTTCTTCTTCTTCTT-CTTTCGGGCTTTCCTTCGACTCAACCGACGACGGTGGCGACTCGTTATTGATCGTATTGG-CCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACCGCTCTTCA-GGAACCCCGGAGATTTGCCCAGAACTCCCCAAAAAACGACTTTCCTCCTTGGCGAGAAGT CGCTCTTCACGC-3 '(SEQ ID NO: 58)

-GCGAGAAGTG 5 '(SEQ ID NO: 57) (SacII cohesive end] (position # 5024 in pAMG22)

01-1718-97 Če

During ligation of the cohesive end of this substituted DNA sequence, the outer AatII and SacII sites are destroyed. There are unique AatIII and SacII sites in the substituted DNA.

B. Human OPG Met [32-401]

In the example, the expression vector used was the vector pAMG21, a derivative of pCFM1656 (ATCC Accession No. 69576), which contained suitable restriction sites for insertion of genes behind the lux PR promoter (a description of the lux expression system can be found in US Patent 5,169,318). The host cell used was GM120 (ATCC accession number 55764). This host has the lacIQ promoter and the lacI gene integrated in the second site of the host chromosome of the prototrophic E. coli K12 host. Other commonly used E. coli expression vectors and host cells are also suitable for expression.

The DNA sequence encoding the N-terminal methionine and amino acids 32-401 of the human OPG polypeptide was introduced under the control of the luxPR promoter into a plasmid expression vector as follows. PCR using oligonucleotides # 1257-20 and # 1257-19 as primers was performed using the matrix plasmid pRcCMV-Hu OPG DNA containing ^{= i =} α "human" ⁱ " ⁼ OPG ~ cDNA,""and" ^ " —Thirty cycle cycles of thirty cycles: each cycle comprising 20 seconds at 94 ° C, 30 seconds at 37 ° C and finally 30 seconds at 72 ° C. The resulting PCR sample was dissolved on an agarose gel, the PCR product was analyzed, purified and restricted with KpnI and BamHI restriction endonuclease and purified. Synthetic oligonucleotides # 1257-21 and # 1257-22 were individually phosphorylated by T4 polynucleotide

01-1718-97 The kinases and ATP and then mixed, heated to 94 ° C and allowed to cool to room temperature to form an oligonucleotide linker duplex containing Nde I and Kpn I cohesive ends. Phosphorylated linker duplex, formed between oligonucleotides # 1257-21 and # 1257-22, containing NdeI and KpnI cohesive ends (see Figure 14A) and KpnI and BamHI digested and purified PCR product, generated using oligo-primers # 1257-20 and # 1257-19 (see above) was directly inserted between the two sites of the plasmid vector pAMG21, namely the NdeI site and the BamHI site, using standard recombinant DNA methodology (see Fig. 14A and sequences below). The synthetic linker used E. coli codons to provide the N-terminal methionine.

Two clones were selected, plasmid DNA was isolated, and the human OPG insert was subsequently confirmed by the DNA sequence. The resulting pAMG21 plasmid containing amino acids 32-401 of human OPG polypeptides immediately following methionine was designated as pAMG21-huOPG met [32-401] or pAMG21-huOPG met [32-401].

Oligo # 1257-19

5 '-TACGCACTGGATCCTTATAAGCAGCTTATTTTTACTGATTGGAC-3' (SEQ ID NO: 59)

Oligo # 1257-20 —- ~ · - = -, 5'-GTCCTCCTGGTACCTACCTAAAACAAC-3 '(SEQ ID NO: 60)

# 1257-21

5 '-TATGGATGAAGAAACTTCTCATCAGCTGCTGTGTGATAAATGTCC

GCCGGGTAC -3 '(SEQ ID NO: 61)

Oligo # 1257-22 ’-CCGGCGGACATTTATCACACAGCAGCTGATGAGAAGTTTCTTCATCCA-3 '(SEQ ID NO: 47)

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Cultures of pAMG21-huOPG met [32-401] in E. coli GM120 in 2XYT medium containing 20 gg / ml kanamycin were incubated at 30 ° C prior to incorporation. Addition of the synthetic autoinducer N- (3-oxohexanoyl) -DL-homoserinlactone to the culture medium to a final concentration of 30 ng / ml achieved induction of huOPG met [32-401] gene production expression from the luxPR promoter and the cultures were incubated either at 30 ° C or 37 ° C for an additional 6 hours. After six hours, the bacterial cultures were examined microscopically to investigate the presence of inclusion bodies and pelleted by centrifugation. The presence of frangible inclusion bodies in induced cultures indicated that part of the recombinant huOPG met [32-401] gene product was produced insoluble in E. coli. Some bacterial pellets were resuspended in 10 mM TrisHCl / pH8, 1 mM EDTA and lysed directly by adding 2X Laemlli sample buffer to 1X final concentration and β-mercaptoethanol to 5% final concentration and analyzed by SDS-PAGE. On an SDS-PAGE gel containing total cell lysates of cultures induced at 30 ° C and 37 ° C, a significantly more intensely stained band (coomassie) of approximately 42 kDa was observed, compared to line 2, which is the total cell lysate of the culture incubated at 30 ° C (Fig. 14E). The expected gene product length should be 370 amino acids and the expected molecular weight is approximately 42.2 kDa. After a six-hour incubation at 37 ° C, another culture was pelleted and further treated by inclusion bodies (see below) either by isolation or by microfluidization. The microfluidization-treated pellet was resuspended in 25 mM TrisHCl / pH8, 0.5 M NaCl buffer with Model 1108 microfluidizer and elongated twenty times (Microfluidics Corp.) to e.

01-1718-97 She gathered. An aliquot was removed from the collected sample (microfluidized total lysate) and the residue pelleted for 20 minutes at 20,000 x g. The resulting supernatant was discarded (microfluidized soluble fraction) and ¹ pellet was resuspended in 25mM Tris-HCl / pH8, 0.5M NaCl, 6M urea solution (microfluidized insoluble fraction). To an aliquot of the total soluble or insoluble fraction, Laemalli sample buffer was added to twice the final concentration and β-mercaptoethanol to 5% of the final concentration. The samples were then analyzed by SDSPAGE. A substantial amount of the recombinant huOPG met [32-401] gene product appears to be present in the insoluble fraction. Recombinant protein inclusion bodies were purified as follows. Bacterial cells were separated from the medium by isopycnic centrifugation in a Beckmann J-6B centrifuge equipped with a JS-4.2 rotor for 15 minutes at 4,900 xg at 4 ° C. The bacterial pellet was resuspended in 5 ml of water and then diluted with water to a final volume of 10 ml. This suspension was transferred to an ice-cooled stainless steel cup and subjected to sonication disruption using a Branson sonicator equipped with a standard end (power setting = 5, duty cycle = 95%, 80 bursts). The sonicated cell suspension was centrifuged for five to ten minutes in a Beckman Optima TLX supercentrifuge equipped with a TLA 100.3 rotor at 195,000 xg at 23 ° C. The supernatant was discarded and the pellet was rinsed with a stream of water from a syringe. The pellets were collected by scraping with a micro spatula and transfected into a glass homogenizer (15 ml capacity). Five ml of Percoll solution (75% liquid Percoll, 0.15 M sodium chloride) was added to the homogenizer and the contents were homogenized until a uniform suspension was obtained.

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The volume was increased by adding Percoll solution to 19.5 ml, mixed and distributed to 3 Beckman Quick-Seal tubes (13 χ 32 mm). The tubes were sealed according to the manufacturer's instructions. The tubes were centrifuged for 30 minutes in a Beckman TLA 100.3 rotor at 23 ° C and 20,000 rpm (21,600 x g). The tubes were analyzed for their respective band pattern. Gradient fractions were isolated and diluted with water to detect brittle bodies. Inclusion bodies were pelleted by centrifugation and protein concentration was determined by SDS-PAGE.

An aliquot of inclusion bodies, isolated as described below, was dissolved in 1X Laemllium sample buffer with 5% β-mercaptoethanol and dissolved on an SDS-PAGE gel, and the isolated inclusion bodies yielded a highly purified recombinant huOPG [32-401] gene product. The main ~ 42 kDa band observed after dissolution of inclusion bodies on the SDS polyacrylamide gel was excised from the separation gel and the N-terminal amino acid sequence was found to be substantially identical to that described by Matsudaira et al., J. Biol. Chem. 262, 10-35 (1987)). The following sequence was determined after 19 cycles:

NH ₂ -MDEETSHQLLCDKCPPGTY-COOH (SEQ ID NO: 62)

7.T -Ϊ <= + Ί 1 that this sequence is identical to the first encoded pAMG21 Huvector produced by nineteen amino acids,

OPG met [32-401] an expression methionine residue that provided a bacterial expression vector.

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C. Human OPG met [22-401]

The DNA sequence encoding the N-terminal methionine and amino acids 22-401 of human OPG was inserted under the control of the luxPR promoter into the prokaryotic plasmid expression vector pAMG21 as follows. The isolated plasmid DNA of pAMG21-huOPG met [32-401] (see Part B) was digested with KpnI and BamHI restriction endonucleases and the resulting fragments were dissolved on an agarose gel. The B fragment (~ 1064 bp fragment) was isolated from the gel using standard methodology. Synthetic oligonucleotides # 1267-06 and # 1267-07 were individually phosphorylated and allowed to form an oligonucleotide linker duplex that contained the NdeI and KpnI cohesive ends, using the procedures in Part B. The synthetic linker used E. coli codons to provide an N-terminal methionine. Phosphorylated linker duplex containing NdeI and KpnI cohesive ends, and the isolated ~ 1064 bp fragment of pAMG21-huOP met [32-401], digested with KpnI and BamHI restriction endonucleases, were directly inserted between the NdeI site and the BamHI site of the plasmid vector using standard recombinant DNA methodology. pAMG21. The ligation mixture was transformed into 393 E. coli host cells by electroporation according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the huOPG-met [22-401] gene.

# 1267-06

5 »-TAT GGA AAC TTT TCC TCC AAA ATA TCT TCA TTA TGA TGA AGA AAC TTC TCA TCA GCT GCT GTG TGA TAA ATG TCC GCC GGG TAC-3 * (SEQ ID NO: 63)

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# 1267-07

5 * -CCG GCG GAT ATT TAT CAC ACA GCA GCT GAT GAG AAG TTT CAT CAT AAT GAA GAT ATT TTG GAA AAG TTT CCA-3 * (SEQ ID NO: 64)

Cultures of pAMG21-huOPG-met [22-401] in E. coli host 393 were plated in 2XYT medium containing 20 gg / ml kanamycin and incubated at 30 ° C prior to induction. Induction of recombinant gene product expression from the luxPR promoter of the pAMG21 vector was achieved by adding a synthetic autoinducer, N- (3-oxohexanoyl) -DL-homoserinlactone, to the culture medium to a final concentration of 30 ng / ml and further incubating at 30 ° C or 37 ° for six hours. C. After six hours, the bacterial cultures were pelleted by centrifugation (= 30 ° C, 1 + 6 or 37 ° C 1 + 6). Bacterial cultures were also pelleted either immediately before induction (= 30 ° C, Prel) or, alternatively, no inducer was added to the separation culture and this culture was incubated at 30 ° C for an additional six hours to give an uninduced (UI) culture ( = 30 [deg.] C.). Bacterial pellets of either 30 ° C Pre I, 30 ° C UI, 30 ° C 1 + 6, or 37 ° C 1 + 6 cultures were resuspended, lysed, and analyzed by SDS-polyacrylamidogel eiektrophoresis (PAGE) under the conditions described in section B. Polyacrylamide gels were either stained with blue coomasia and / or Western blotted to nitrocellulose and immunosonded with rabbit anti-OPG-Fc polyclonal antibody as described in Example 10. The level of gene product resulting from induction was compared to uninduced (30 ° C UI) ) or a pre-induced (30 ° C Pre) sample.

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D. Mouse OPG met [22-401]

N-terminal methionine and (mu) OPG polypeptide, se

The DNA sequence encoding amino acids 22-401 of the mouse was inserted into the prokaryotic plasmid expression vector pAMG21 under the control of the luxPR promoter as follows. PCR using oligonucleotides # 1257-16 and # 1257-15 as primers was performed using the pRcCMV-Mu OPG DNA plasmid and the thermocyclic conditions described in section B. The resulting PCR product was digested with KpnI and BamHI restriction endonuclease as described in section B. Synthetic Oligonucleotides # 1260-61 and # 1260-82 were individually phosphorylated and allowed to form an oligonucleotide linker duplex that contained NdeI and KpnI cohesive ends using the procedures in Section B. The synthetic linker used E. coli codons to yield methionine. Phosphorylated linker duplex, oligonucleotides # 1260-61 and # 1260-82, containing NdeI and KpnI cohesive ends and digested with KpnI and BamHI restriction endonuclease, and purified PCR product generated using oligonucleotide primers # 1257-16 and # 1257-15 using standard recombinant DNA methodologies directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21. The ligation mixture was transformed into host cells by electroporation. E. coli procedure recommended by the manufacturer. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG-met [22-401] gene.

N-terminal created

Expression of recombinant muOPG met [22-401] polypeptide from 393 cell cultures containing plasmid pAMG21-MuOPG met [22-401] following induction was determined using the procedures described in section C.

• · · · · · ·

01-1718-97 English ·· ··

# 1257-15

5'-TAC GCA GAT GAT CCT TAT AAG CAG GAT TTC ACG GAT TGA AC-3 '(SEQ ID NO: 65)

Oligo # 1257-16 .......

'-GTG CTC CTG GTA CCT ACC TAA AAC AGC ACT GCA CAG TG-3' (SEQ ID NO: 66)

# 1260-61

5 '-TAT GGA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GCT GTG TGA TAA ATG TGC TCC GGG TAC-3' (SEQ ID NO: 67)

# 1260-82

5 ¹ -CCG GAG GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT TTT CCA3 '(SEQ ID NO: 68)

E. Mouse OPG met [32-401]

The DNA sequence encoding the N-terminal methionine and amino acids 32-401 of the mouse OPG polypeptide was inserted into the prokaryotic plasmid expression vector pAMG21 as follows, under the control of the luxPR promoter. Synthetic oligonucleotides # 1267-08 and # 1267-09 were individually phosphorylated and allowed to form an "oligonucleotide spoj" linker duplex using the procedures described in "Part B". The synthetic linker duplex used E. coli codons to provide the N-terminal methionine. Phosphorylated linker duplex, formed between oligonucleotides # 126708 and # 1267-09, containing NdeI and KpnI cohesive ends and digested with KpnI and BamHI restriction endonucleases, and the purified PCR product described previously (part D), directly using standard recombinant DNA methodology

01-1718-97 English ····

Placed an NdeI site and a BamHI site of the plasmid vector pAMG21. The ligation mixture was transformed into E. coli host 393 cells by electroporation according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG-met [32-401] gene.

Expression of recombinant muOPG met [32-401] polypeptides from 393 cell cultures containing the recombinant plasmid pAMG21 due to induction was determined using the procedures described in section C.

# 1267-08

5 '-TAT GGA CCC AGA AAC TGG TCA GCT GCT GTG TGA TAA ATG TGC TCC GGG TAC-3' (SEQ ID NO: 69)

# 1267-09

5'-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CTG GGT CCA-3 '(SEQ ID NO: 70)

F. mouse OPG met-lys [22-401]

The DNA sequence encoding the N-terminal methionine followed by the lysine residue and amino acids 22-401 of mouse OPG was inserted into the prokaryotic plasmid expression vector pAMG21 as follows, under the control of the luxPR promoter. Synthetic oligonucleotides # 1282-95 and # 1282-96 were individually phosphorylated and allowed to form an oligonucleotide linker duplex using the procedures described in Part B. The synthetic linker duplex used E. coli codons to provide the N-terminal methionine. Phosphorylated linker duplex, formed between oligonucleotides # 128201-1718-97 Če ·· · · · · ···

999 · 9

99 and # 1282-96, containing the NdeI and KpnI cohesive ends and digested with KpnI and BamHI restriction endonucleases, and the purified PCR product described previously (part D), were directly inserted between the NdeI site and the BamHI site of the plasmid using standard recombinant DNA methodology. vector pAMG21. The ligation mixture was transformed into E. coli host 3 93 cells by electroporation according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG-Met-Lys [22-401] gene.

Expression of recombinant muOPG Met-Lys [32-401] polypeptides from 393 cell cultures containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

# 1282-95

5'-TAT GAA AAC TCT GCC TCC AAA ATA CCT GCA TTA CGA TCC GGA AAC TGG TCA TCA GCT GTG TGA TAA ATG TGC TCC GGG TAC-3 '(SEQ ID NO: 71)

# 1282-96

5 '-CCG GAG GAT GAT GAT GAT GG GAT GAT GG GAT GAT GAT GG GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT GAT

G. Mouse OPG met-lys- (his) 7 [22-401]

The DNA sequence encoding the N-terminal Met-Lys-HisHis-His-His-His-His-His (= MKH) residues followed by amino acids 22-401 of mouse OPG was inserted into the prokaryotic plasmid expression expression under the control of the luxPR promoter.

01-1718-97 pAMG21 vector as follows. PCR using oligonucleotides # 1300-50 and # 1257-15 as primers was performed using the pAMG21-muOPGmet [22-401] DNA plasmid and the thermocyclic conditions described in section B. The resulting PCR sample was dissolved on an agarose gel, PCR the product was excised, purified, digested with NdeI and BamHI restriction endonuclease and purified. The NdeI and BamHI digested and purified PCR product, generated using oligonucleotide primers # 1300-50 and # 1257-15, were directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21 using standard recombinant DNA methodology. The ligation mixture was transformed into E. coli host 393 cells by electroporation according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the MuOPG-MKH [22-401] gene.

Expression of recombinant muOPG MKH [22-401] polypeptide from transformed 393 cultures containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

# 1300-50

5'-GTT CTC CTC ATA TGA AAC ATC ATC ACC ATC ACC ATC ATG AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG AT-3 '(SEQ ID NO: 73)

Oligo # 1257-15 (see section D) • ·

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H. Mouse OPG met-lys [22-401] (his) ₇

The DNA sequence encoding the N-terminal met-lys, amino acids 22 through 401 murine OPG, and seven histamine residues following amino acid 401 (= muOPG MK [22-401] H _7), was placed under control of the lux PR promoter in prokaryotic expression vector pAMG21 as follows way. PCR using oligonucleotides # 1300-49 and # 1257-51 as primers was performed using the pAMG21-muOPG-met [22-401] DNA plasmid and the thermocyclic conditions described in Part B. The resulting PCR sample was dissolved on an agarose gel, The PCR product was purified, digested with endonuclease, and purified. and the purified PCR product, using oligonucleotide primers # 1300-50 and # 1257-15, was directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21 using standard recombinant DNA methodology. Ligation was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-MK [22-401] -H ₇ gene.

Ndel and BamHI

NdeI and BamHI generated to excise, restriction digested _____________ F.vprpRn-. of the mutant = muOPG = =22-401] -H ₇ polypeptide from transformed 393 cultures containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

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# 1300-49

5'-GTT CTC CTC ATA TGA AAG AAA CTC TGC CTC ACA ACC TGC A-3 '(SEQ ID NO: 74)

# 1300-51

5'-TAC GCA CTG GAT CCT TAA TGA TGG TGA TGG TGA TGA TGT AAG CAG CTT ATT TTC ACG GAT TGA ACC TGA TTC CCT A-3 '(SEQ ID NO: 75)

I. Mouse OPG met [27-401]

The DNA sequence encoding the N-terminal methionine and amino acids 27-401 of mouse OPG was placed under the control of the luxPR promoter in the prokaryotic plasmid expression vector pAMG21 as follows. PCR using oligonucleotides # 1309-74 and # 1257-15 as primers was performed using the pAMG21-muOPG-met [22-401] DNA plasmid and the thermocyclic conditions described in Part B. The resulting PCR sample was dissolved on an agarose gel, The PCR product was isolated, purified, digested with NdeI and BamHI restriction endonuclease and purified. The NdeI and BamHI digested and purified PCR product were using standard recombinant DNA methodology

ranged, directly between the NdeI site, and the BamHI site, the "plasmid site" of pAMG21. Ligation was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [27-401] gene.

Expression of recombinant muOPG-met [27-401] polypeptide from transformed 393 cultures they contained

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the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

# 1309-74

5'-GTT CTC CTC ATA TGA AAT ACC TGC ATT ACG ATC CGG AAA CTG GTC AT-3 '(SEQ ID NO: 76)

Oligo # 1257-15 (see Section D)

J. Humane OPG met [27—401]

The DNA sequence encoding the N-terminal methionine and amino acids 27-401 of human OPG was placed under the control of the luxPR promoter in the prokaryotic plasmid expression vector pAMG21 as follows. PCR using oligonucleotides # 1309-75 and # 1309-76 as primers was performed using the pAMG21-huOPG-met [22-401] DNA plasmid and the thermocyclic conditions described in Part B. The resulting PCR sample was dissolved on an agarose gel, The PCR product was isolated, purified, digested with Asel and BamHI restriction endonuclease and purified. The Asel and BamHI digested and purified PCR product were directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21 using standard recombinant DNA methodology. The ligation mixture was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the huOPG-met [27-401] gene.

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Expression of recombinant huOPG-met [27-401] polypeptide from transformed 393 cells containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

# 1309-75

5'-GTT CTC CTA TTA ATG AAA TAT CTT CAT GAT GAA GAA ACT T-3 '(SEQ ID NO: 77)

# 1309-76

5 * -TAC GCA CTG GAT CAT TAT AAG CAG CTT ATT TTT ACT GAT T-3 '(SEQ ID NO: 78)

K. Mouse OPG met [22-180]

The DNA sequence encoding the N-terminal methionine and amino acids 22-180 of mouse OPG was placed under the control of the luxPR promoter into the prokaryotic plasmid expression vector pAMG21 as follows. PCR using oligonucleotides # 1309-72 and # 1309-73 as primers was performed using the pAMG21-muOPGmet [22-401] DNA plasmid and the thermocyclic conditions described in Part B. The resulting PCR sample was dissolved on an agarose gel, PCR. the product was isolated, purified, digested with NdeI and BamHI restriction endonuclease and purified. The NdeI and BamHI digested and purified PCR product was directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21 using standard recombinant DNA methodology. The ligation was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated and performed

01-1718-97 • ♦ ♦ ♦ »» »» »»

DNA sequencing to verify the DNA sequence of the muOPG-met [22-180] gene.

The expression of recombinant muOPG-met [22-180] polypeptides from transformed 393 cultures containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

# 1309-72

5'-GTT CTC CTC ATA TGG AAA CTC TGC CTC CAA AAT ACC TGC A-3 '(SEQ ID NO: 79)

# 1309-73

5 '-TAC GCA CTG GAT CCT TAT GTT GCA TTT CCT TTC TGA ATT AGC A-3'

L. Mouse OPG met [27-180]

The DNA sequence encoding the N-terminal methionine and amino acids 27-180 of mouse OPG was placed under the control of the luxPR promoter into the prokaryotic plasmid expression vector pAMG21 as follows. PCR using oligonucleotides # 1309-74 (see Part I) and # 1309-73 (see Part K) as primers was performed using the pAMG21-muOPG-met [22-401] DNA plasmid matrix and the thermocyclic conditions described in Part B The resulting PCR sample was dissolved on an agarose gel, the PCR product was removed, purified, digested with NdeI and BamHI restriction endonuclease and purified. The above NdeI and BamHI digested and purified PCR product was directly inserted between the NdeI site and the BamHI site of the plasmid vector pAMG21 using standard recombinant DNA methodology. Ligace se

393 E. coli cells selected, isolated

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100 electroporations were transferred to the host by the manufacturer's recommended procedure. The clones were plasmid DNA and DNA sequenced to verify the DNA sequence of the muOPG-met [27-180] gene.

Expression of recombinant muOPG met [27-180] polypeptides from transformed 393 cultures containing the recombinant plasmid pAMG21 following induction was determined using the procedures described in section C.

M. Mouse OPG met [22-189] and met [22-194]

The DNA sequence encoding the N-terminal methionine and either amino acids 22-189 or 22-194 of mouse OPG was placed under the control of the luxPR promoter into the prokaryotic expression vector pAMG21 as follows. A pair of synthetic oligonucleotides # 1337-92 and # 1337-93 (= muOPG-189 linker) or # 1333-57 and # 1333-58 (= muOPG-194 linker) were individually phosphorylated and allowed to form an oligonucleotide duplex pair using the methods described The purified plasmid DNA of pAMG21-muOPG-met [22-401] was digested with KpnI and BspEI restriction endonucleases and the resulting DNA fragments were dissolved on an agarose gel. An approximately 413 bp B fragment was isolated using standard recombinant DNA methodology. Phosphorylated oligonucleotide duplexes were formed either between oligonucleotides # 1337-92 and # 13337-93 (muOPG-189 linker) or between oligonucleotides # 1333-57 and # 1333-58 (muOPG-194 linker) containing BspEI and BamHI cohesive ends, and above said isolated ~ 413 bp B fragment of plasmid pAMG21-muOPGmet [22-401], digested with KpnI and BspEI restriction τ dr

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101 endonucleases were directly inserted between the KpnI site and the BamHI site of pAMG21-muOPG met using standard recombinant DNA methodology [22-401]. Each ligation mixture was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [22-189] gene or the muOPG-met [22-194] gene.

Expression of recombinant muOPG-met [22-189] and muOPGmet [22-194] polypeptides from recombinant pAMG21 plasmids transformed into 393 cells was detected using the procedures described in section C.

# 1337-92

5'-CCG GAA ACA GAT AAT GAG-3 '(SEQ ID NO: 81)

# 1337-93

5'-GAT CCT CAT TAT CTG TTT-3 '(SEQ ID NO: 82)

# 1333-57

5'-CCG GAA ACA GAG AAG CGCA AAA AGT AAG-3 '(SEQ ID NO: 83)

# 1333-58

5'-GAT TCT TTT TGC GTG GCT TCT CTT TTT-3 '(SEO ID NO: 84). ..

N. Mouse met [27-189] and met [27-194]

The DNA sequence encoding the N-terminal methionine and either amino acids 27-189 or 27-194 of mouse OPG was placed under the control of the luxPR promoter into the prokaryotic expression vector pAMG21 as follows. Phosphorylated oligonucleotide linkers, "muOPG-189 linker" or "muOPG-194 linker" (see Part M) containing BspEI and BamHI e 9

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The 102 cohesive ends and the isolated -413 bp B fragment of plasmid pAMG21-muOPG-met [22-401], digested with KpnI and BspEI restriction endonucleases, were directly inserted between the KpnI site and the BamHI site of pAMG21-muOPG met [11] using standard recombinant DNA methodology. 27-401]. Each ligation mixture was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the muOPG-met [27-189] gene or the muOPG-met [27-194] gene.

Expression of recombinant muOPG-met [27-189] and muOPGmet [27-194] polypeptides from recombinant pAMG21 plasmids transformed into 393 cells was detected using the procedures described in section C.

0. Human OPG met [22-185], met [22-189], met [22-194]

The DNA sequence encoding the N-terminal methionine and either amino acids 22-185, 22-189 or 22-194 of human OPG was placed under the control of the luxPR promoter into the prokaryotic expression vector pAMG21 as follows. Synthetic oligonucleotide pair, # 1331-87 and # 1331-88 (= huOPG-185 linker), # 1331-89 and # 1331-90 (= huOPG-189 linker) or # 1331-91 and # 1331-92 (= huOPG -194 linker), were individually phosphorylated and individually allowed to form an oligo-linker duplex pair using the methods described in section B. The purified plasmid DNA of pAMG21-huOPG-met [27-401] was digested with KpnI and NdeI restriction endonucleases and the resulting DNA fragments. were dissolved on an agarose gel. An approximately 407 bp B fragment was isolated using standard recombinant DNA standard

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«· ··

103 # 1331-90 (= huOPG-189 (huOPG-194 linker) methodology) Phosphorylated oligo-linker duplexes, formed either between oligonucleotides # 1331-87 and # 1331-88 (= huOPG-185 linker), # 1331-89 and or # 1331-91 and # 1331-92 [each linker containing NdeI and BamHI cohesive ends], and the above-isolated isolated ~ 407 bp B fragment of plasmid pAMG21-huOPG-met [27-401], digested with KpnI and NdeI restriction. endonucleases, were directly inserted between the KpnI site and the BamHI site of plasmid pAMG21-huOPG met using standard recombinant DNA methodology [22-401]. Each ligation was electroporated into 393 E. coli host cells according to the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated, and DNA sequencing was performed to verify the DNA sequence of the huOPG-met [22-185] gene, huOPG-met [22-189] gene, or huOPG-met [22-194] gene.

Expression of recombinant huOPG-met [22-185], huOPGmet [22-189] or huOPG-met [22-194] in transformed 393 cells containing recombinant pAMG21 plasmids was detected using the procedures described in section C.

# 1331-87

5'-TAT GTT AAT GAG-3 '(SEQ ID NO: 85)

# 1331-88

5'-GAT CCT CAT TAA CA-3 '(SEQ ID NO: 86)

# 1331-89

5'-TAT GTT CCG GAA ACA GTT AAG-3 '(SEQ ID NO: 87)

# 1331-90

5'-GAT CCT TAA CTG TTT CCG GAA CA-3 '(SEQ ID NO: 88)

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CAA CTC AAA AAT

TCA CTG TTT CCG GAA ^:

104

# 1331-91

5'-TAT GTT CCG GAA ACA GTG AAT AAG-3 '(SEQ ID NO: 89)

# 1331-92

5 '-GAT CCT TAT TTT TGA GTT GAT CA-3' (SEQ ID NO: 90)

P. Human OPG met [27-185], met [27-189], met [27-194]

The DNA sequence encoding the N-terminal methionine and either amino acids 27 to 185, 27 to 189 or 27 to 194 of human OPG polypeptides was placed under the control of the luxPR promoter into the prokaryotic expression vector pAMG21 as follows. Phosphorylated oligonucleotide linkers "huOPG-185 linker," huOPG-189 linker, or "huOPG194 linker" (see Part O) containing the NdeI and BamHI cohesive ends and the isolated ~ 407 bp B fragment of plasmid pAMG21huOPG-met [27-401], digested with KpnI and NdeI restriction endonucleases (see section O) were directly inserted between the KpnI site and the BamHI site of the plasmid pAMG21-huOPG met [27-401] using standard recombinant DNA methodology (see section J). Each ligation mixture was transferred to host 393 E cells by electroporation, following the manufacturer's recommended procedure. Clones were selected, plasmid DNA was isolated and subjected to DNA jsekvencování ₌ intended to verify _DNA huOPGmet sequence [27-185] huOPGmet [27-189] or huOPGmet [27-194] gene.

The expression of recombinant huOPG-met [27-185], huOPGmet [27-189] and huOPG-met [27-194] polypeptides from recombinant pAMG21 plasmids transformed into 393 cultures was detected using the procedures described in section C.

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Q. Mouse OPG met [27-401] (P33E, G36S, A45P)

The DNA sequence encoding the N-terminal methionine and either amino acids 27-48 of human and subsequently amino acid residues 49-40 of murine OPG was put under control. TheuxPR promoter into the prokaryotic expression vector pAMG21 as follows. The purified plasmid pAMG21-huOPGmet [27-401] (see section J) was digested with AatII and KpnI restriction endonucleases and the ~ 1075 bp B fragment isolated from the agarose gel was isolated from the agarose gel using standard recombinant DNA methodology. In addition, plasmid pAMG21-muOPG-met [22-401] DNA (see section D) was digested with KpnI and BamHI restriction endonucleases and the isolated -1064 bp B fragment described above. Isolated ~ 1075 bp pAMG21-huOPG-met [27-401] restriction fragment containing AatII & KpnI cohesive ends (see above), -1064 bp pAMG21-muOPG-met [22-401] restriction fragment containing KpnI and BamHI cohesive ends and - The 5043 bp restriction fragment containing the AatII and BamHI cohesive ends and the corresponding pAMG21 nucleic acid sequence between AatII & BamHI were ligated using standard recombinant DNA methodology. The ligation was electroporated into E. coli host 393 cells according to the manufacturer's recommended procedure. Clones were selected and the presence of the recombinant insert in the plasmid was verified using standard DNA methodology. The amino acid changes in muOPG from proline-33 to glutamic acid-33, from glycine-36 to serine-36, and from alanine-45 to proline-45 result from the replacement of muOPG residues 27-48 of huOPG residues 27-48.

Recombinant muOPG-met expression [27-401] (P33E, G36S,

A45P) from transformed 393 cells containing

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The 10 6 recombinant pAMG21 plasmid was determined by the methods described in Part C.

R. Mouse OPG met-lys (his) 2-ala-ser- (asp) 4-lys [22-401] (A45T)

DNA sequence encoding an N-terminal His and an enterokinase recognizing a sequence that is (from NH ₂ to the COOH end) Met-Lys-His-His-His-His-His-His-His-His-Ala-Ser-Asp-Asp-AspAsp -Lys (= HEK), followed by amino acids 22-401 of the mouse OPG polypeptide, was placed under the control of the lac repressor regulated by the Ps4 promoter as follows. pAMG22-His (see Part A) was digested with NheI and BamHI restriction endonucleases and the large fragment (fragment A) was isolated from an agarose gel using standard recombinant DNA methodology. Oligonucleotides # 1282-91 and # 1282-92 were individually phosphorylated and allowed to form an oligonucleotide duplex using the methods described above (see Part B). Phosphorylated binding duplex, formed between oligonucleotides # 1282-91 and # 1282-92, containing the Nhel and KpnI cohesive ends, described by the KpnI and BamHI digested and purified PCR product (see section D), and the A fragment of pAMG22-His vector digested with NheI and BamHI were ligated using standard recombinant DNA methodology. Ligacs were transformed into E. coli host GM120 by electroporation carried out in the manner recommended by the manufacturer. Clones were selected, plasmid DNA was isolated, and DNA sequencing performed to verify the DNA sequence of the muOPG-HEK [22-401] gene. DNA sequencing revealed a non-native mutation in the natural muOPG sequence that resulted from the change of a single amino acid of alanine-45 muOPG polypeptides to threonine.

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107

The expression of recombinant muOPG-HEK [22-401] (A45T) from GM120 cells containing the recombinant pAMG21 plasmid was determined using methods essentially identical to those described in Part C except that instead of adding a synthetic autoinductor to achieve induction, IPTG is added to the final concentration 0.4 mM.

# 1282-91

5 * -CTA GCG ACG ACG ACG ACA AAA CTC TGC CTC CAA AAT ACC TGC ATT ACG ATC CGG AAA CTG GTC ATC AGC TGC TGT GTG ATA AAT GTG CTC CGG GTA C-3 ´ (SEQ ID NO: 91)

Oligo # 1282-92 '-CCG GAG CAC ATT TAT CAC ACA GCA GCT GAT GAC CAG TTT CCG GAT CGT AAT GCA GGT ATT TTG GAG GCA TTT CTT TGT CGT CGT CGT CG-3' (SEQ ID NO: 92): S . Human OPG met-arg-gly-ser- (his) ₆ [22-401]

Eight oligonucleotides (1338-09 to 1338-16, listed below) were designed to prepare a 175 base fragment for overlapping double stranded DNA. These oligonucleotides are conditioned, ligated, and the 5 'and 3' oligos were used as PCR primers for the preparation -Greater mnoŽ8tví ⁼ ~ "f ragmentu composed of" 175 "" bases. The final PCR gene products were digested with restriction endonucleases Clal and KpnI to yield a fragment that replaced the 28 N-terminal codons of human OPG. The ClaI and KpnI digested PCR product was inserted into pAMG21-huOPG [27-401], which was also digested with ClaI and KpnI. The ligated DNA was transformed into competent E. coli 393 host cells. Clones were screened to determine if they were able to.

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108 to produce a recombinant protein product and whether they contain a gene fusion having the correct nucleotide sequence. Protein expression levels were determined based on studies performed in a 50 ml shaker. The whole cell lysate and sonised pellet were analyzed for expression of the construct using stained Coomassie stained PAGE gels and Western analysis using a mouse anti-OPG antibody. The expression of huOPG Met-Arg-Gly-Ser- (His) _s [22-401], resulting in the formation of large inclusion bodies and protein, was localized to the insoluble (pellet) fraction.

1338-09

ACA AAC ACA ATA GAT TTG ATA CTA GA (SEQ ID NO: 93)

1338-10

TTT GTT TAT ACT AAT TAA AGG AAT AAA ATA TGA GAG GAT CGC ATC AC (SEQ ID NO: 94)

1338-11

CAT CAC CAT CAC GA TAC CC TAC CCG TAC GAC TAC TAC GAC TAC GAC GAA GA (SEQ ID NO: 95)

1338-12

AAC CTC CCA CCT GCT GCT GTG CGA CAA ATG CCC GCC GGG TAC CCA AAC A (SEQ ID NO: 96)

1338-13

TGG TTG GGT ACC GGG GGT GCA TTT GT (SEQ ID NO: 97)

1338-14

CGC ACA GCA GCT GGT GGG AGT TTT CTT CGT CGT GCC GGT ATT TCG GC (SEQ ID NO: 98)

1338-15

GGG AAG GTT TG TGA TGG TGA TGG TGA TGC GAT CCT ATA TTT TAT T (SEQ ID NO: 99)

1338-16

CCT TCT ATT. AGT TAA AAC AAA TCT AGT-ATC AAA TCG ATT GTG TTT GT ---------- (SEQ ID NO: 100)

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T. Human OPG met-lys [22-401] and met (lys) ₃ [22-401]

To construct the met-lys and met- (lys) ₃ versions of human OPG [22-401], overlapping oligonucleotides were designed to add the appropriate number of lysine residues. Two oligonucleotides were designed for each construct so that the overlap allowed two cycles of PCR to produce the final product. The matrix for the first PCR reaction was a plasmid DNA preparation containing the human 22-401 gene. The first PCR added the lysine residue (s). The second PCR used the first cycle product and added the sequence back to the first restriction site, Clal.

The final PCR gene products were digested with restriction endonucleases Clal and KpnI, which replaced the N-terminal 28 codons of hu OPG and subsequently ligated into plasmid pAMG21-hu OPG [27-401], which was also digested with the two restriction endonucleases. Ligated DNA was transformed into competent host cells of E. coli strain 393. Screening of the clones revealed their ability to produce a recombinant protein product and whether they showed gene fusion with the correct nucleotide sequence. Protein expression levels were determined from 50 ml shaker studies. Whole cell lysate and sonicated pellet were analyzed for expression of the construct using coomassie-stained PAGE gels and western analysis with mouse anti-OPG antibody. No constructs detected detectable levels of protein expression or observed inclusion bodies. DNA sequences were confirmed by DNA sequencing.

The following oligonucleotide primers were used to prepare Met-Lys huOPG [22-401]:

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TTT TAA CTA ATT

C (SEQ ID NO

110

1338-17

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 101)

1338-18

ATA AAA GGA ATA AAA ATA AAA ATA AAA ATA (SEQ ID NO: 102)

1338-20

TGT TTG GGT ACC CGG CGG ACA

Oligonucleotide primers to prepare Met- (Lys) 3-huOPG [22401]:

1338-17

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 104)

1338-19

ATA AAA GGA ATA AAA ATA AAA ATA AAA ATA AAA ATA AAA ATA AAA ATA (SEQ ID NO: 105)

1338-20

TGT TTG GGT ACC GGT CGG ACA TTT ATC ACA C (SEQ ID NO: 106)

CTT TTC CTC CAA

U. Fusion of human and mouse OPG [22-4011 / Fc

Four OPG-Fc fusions were constructed in which the Fc region of human IgG1 bound to the N-terminus of human or mouse osteoprotegerin amino acids 22-401 (designated Fc / OPG [22-401]) or the C-terminus (designated OPG [22]). -401] (Fc). Fc fusions were constructed using the pFc-A3 fusion vector described in Example 7.

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All fusion genes were constructed using standard PCR technology. The matrix for PCR reactions was plasinide preparations containing target genes. Overlapping oligos were designed to combine ^one C-terminal segment of the gene with the N terminal portion of another gene. This method allows two genes to be fused together in the correct reading frame after appropriate PCR reactions have been performed. First, a universal fusion oligonucleotide for each gene carrying the target gene was introduced into the PCR reaction. Then, a second complementary fusion oligonucleotide was introduced into the PCR reaction using universal primers to obtain a second gene. At the end of the first PCR reaction, two separate products were obtained, with a fusion site present in each individual gene, providing sufficient overlap to carry out the second cycle of PCR and generate the desired fusion. In the second PCR cycle, the first two PCR products were combined with the universal primers and a full-length fusion DNA sequence was prepared by overlapping regions.

The final PCR gene products were digested with restriction endonucleases XbaI and BamHI and subsequently ligated into the vector pAMG21, which was also digested with the two endonucleases. Ligated DNA was transformed into competent host cells of E. coli bacterial strain 393. The clones were screened for their ability to produce a recombinant protein product and whether they showed gene fusion with the correct nucleotide sequence. Protein expression levels were determined from 50-ml shaker studies. Whole cell lysate and sonicated pellet were analyzed to determine expression of the construct

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112 using coomassie-stained PAGE gels and Western analysis with a mouse anti-OPG antibody.

Fc / huOPG [22-401]

Fc / hu OPG [22-401] fusion peptide expression was detected on a coomassie-stained PAGE gel and western blot. The cells had very large inclusion bodies and much of the product was in the insoluble (pellet) fraction. The following primers were used to construct this OPG-Fc fusion:

1318-48

CAG CCC GGG TAA AAT GGA AAC GTT TCC TCA AAA ATA TCT TCA TT (SEQ ID NO: 107)

1318-49

CGT TTC CAT TTT ACC CGG GCT CAG GAG CCT GC CTT CCT GC GT (SEQ ID NO: 108)

Fc / muOPG [22-401]

Expression of the fusion peptides was detected on a coomassie-stained PAGE gel and western blot. The cells had very large inclusion bodies and much of the product was in the insoluble (pellet) fraction. The following primers were used to construct this OPG-Fc fusion:

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1318-50

CGC TCA GCC GGGTA GTA AAA TGG AAA CGT TGC CTC CAA AAT ACC TGC (SEQ ID NO: 109)

1318-51

CCA TTT TAC CCG GGC TGA GCG AGA GGC TCT TCT GCG TGT (SEQ ID NO: 110) muOPG [22-401] / Fc

Expression of the fusion peptide was detected on a coomassie-stained PAGE gel and western blot. The amount of recombinant product was less than OPG fusion proteins having the Fc region at the N-terminal position. Obvious inclusion bodies were not detected. Most of the product appeared to be in the insoluble (pellet) form. The following primers were used to construct this OPG-Fc fusion:

1318-54

GAA AAT AAG CTG CTT AGC TGC TGA ACC AAA ATC (SEQ ID NO: 111)

1318-55

CAG AGG AGAG AGAG AGAG AGAG AGAG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG

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114 huOPG [22-401] / Fc

Expression of the fusion peptide was not detected on the coomassie-stained gel, although a weak positive signal was seen on the western blot. Obvious inclusion bodies were not detected. The following primers were used to prepare this OPG-Fc fusion:

1318-52

AAA AAT AAG CTG CTG AGC TGC AGC TGA ACC AAA ATC (SEQ ID NO: 113)

1318-53

CAG AGG AGAG AGAG AGAG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG AG

V. Fusion of human OPG met [22-401] -Fc (P25A)

This construct combines the amino acid change of proline to alanine at position 25 (P25A) with huOPG met [22-401] -Fc fusion. The plasmid was digested with the restriction endonucleases Clal and KpnI, which removed the 28 N-terminal codons of the gene and the resulting small (shorter than 200 base pairs) fragment was cut into the Toni-Framrmpn. · Ť η Α ΠΊ sťn

W --------------------, ------- _u -------------- ------ The proline alanine was then ligated into plasmid pAMG21huOPG [22-401] -Fc fusion, which was digested with the two restriction endonucleases. Ligated DNA was transformed into competent host cells of E. coli bacterial strain 393. The clones were screened for their ability to produce a recombinant protein product and whether they showed gene fusion with the correct nucleotide

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115 sequences. Protein expression levels were determined from ml shaker studies. Whole cell lysate and sonicated pellet were analyzed for expression of the construct using coomassie-stained PAGE gels and western analysis with mouse anti-OPG antibody. The expression level of the fusion peptide was detected on a coomassage-stained gel and western blot. The protein was found in the insoluble (pellet) fraction. The cells had large inclusion bodies.

W. Human OPG met [22-401] (P25A)

The DNA sequence encoding the N-terminal methionine and amino acids 22-401 of human OPG with alanine-substituted proline at position 25 under the control of the lux P _R promoter in the prokaryotic expression vector pAMG21 was constructed as follows. Synthetic oligonucleotides # 1289-84 and 1289-85 were annealed to form an oligonucleotide linker duplex with XbaI and KpnI cohesive ends. The synthetic linker duplex used optimal E. coli codons and encoded the N-terminal methionine. The binding also contained a SpeI restriction site that was not included in the original sequence. Binding duplex was routed between XbaI and KpnI using standard methods instead of pAMG21-huOPG-22-401. The ligation ₌ mixture was introduced into E. coli host GM221 by transformation. The clones were first screened for recombinant protein production. Plasmid DNA was isolated from positive clones and subjected to DNA sequencing to verify the DNA sequence of the HuOPG-Met [22-401] (P25A) gene. The following oligonucleotides were used to generate the XbaI-KpnI linker:

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116

# 1289-84

5 '-CTA GAA GGA ATA ACA TAT GGA AAC TTT TGC TCC

AAA ATA TCT TCA TCT TCT GCT GCT

GTG TGA TAA ATG-TCC GCC GGG TAC-3 * (SEQ ID NO: 115)

# 1289-85

5'- CCG GC GAT ATT TAT CAC ACA GAT GAT GAT TAT TTT CTT CAT CAT AAT GAA GATAT ATT TTG GAT CAA AAG TTT CAT GTAT ATT CCT TCT-3 '(SEQ ID NO: 116)

X. Human OPG met [22-401] (P26A) and (P26D)

The DNA sequence encoding the N-terminal methionine and amino acids 22-401 of human OPG with alanine substituted proline at position 26 under the control of the lux P _R promoter in the prokaryotic expression vector pAMG21 was constructed as follows. Synthetic oligonucleotides # 1289-86 and 1289-87 were annealed to form an oligonucleotide linker duplex with XbaI and SpeI cohesive ends. The synthetic linker duplex used optimal E. coli codons and encoded the N-terminal methionine. Binding duplex was routed between XbaI and SpeI using standard methods instead of pAMG21-huOPG [22-401] (P25A). The ligation mixture was transformed into E. coli host GM221 by transformation. The clones were first screened for recombinant protein production. Plasmid DNA was isolated from positive clones and subjected to DNA sequencing to verify the DNA sequence of the huOPG-Met [22-401] (P26A) gene. One of the sequenced clones has been shown to have proline at position 26 substituted with aspartic acid rather than alanine, and this clone has been designated huOPG-met [22 ··]

I 4 ·· · '·

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117

401] (P26D). The following oligonucleotides were used to generate the XbaI-SpeI linker:

# 1289-86

5 ’- CTA GAA GGA GGA ATA ACA TAT GGA AAC TTT TCC TGC TAA ATA TCT TCA TGA TGA AGGA AA - 3 '(SEQ ID NO: 117)

# 1289-87

5 '- CTA GTT TCA TCA TCA TAA TGA AGA TAT GTA GCA AAA GTT TCC ATA TGT TAT TCC TT - 3' (SEQ ID NO: 118)

Y. Human OPG met [22-194] (P25A)

The DNA sequence encoding the N-terminal methionine and amino acids 22 to 194 of human OPG with alanine-substituted proline at position 25 under the control of the lux P _R promoter in the prokaryotic expression vector pAMG21 was constructed as follows. Both plasmid pAMG21-huOPG [27-194] and pAMG21-huOPG [22-401] (P25A) were digested with KpnI and BamHI endonuclease. The 450 bp fragment was isolated from pAMG21huOPG [27-194] and ____6. 1, kbp fragment. was isolated from pAMG21huOPG [22-401] (P25A). These fragments were ligated with each other by transformation and introduced into E. coli host GM221. The clones were first screened for recombinant protein production. Plasmid DNA was isolated from positive clones, and the DNA sequence of the huOPG-Met [22-194] (P25A) gene was verified by DNA sequencing.

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Example 9

Coupling of OPG monomers

CHO cells artificially prepared for overexpressing muOPG [22-401] were used to generate conditioned media for the purpose of analysis of secreted recombinant OPG performed using rabbit polyclonal anti-OPG antibodies. An aliquot of the conditioned medium was concentrated 20 times and then analyzed by reducing and non-reducing SDS-PAGE (Fig. 15). Under reducing conditions, the protein migrated as a Mr 50-55 kd polypeptide. This behavior could be expected if the mature product were glycosylated at one or more of its conventional N-linked glycosylation sites. A surprising finding was made when the sample was analyzed by non-reducing SDS-PAGE. In this case, the protein migrated as approximately 100 kd polypeptide, which is twice the size of the reduced protein. In addition, there was a small amount of Mr 50-55 kd polypeptides. This SDS-PAGE migration pattern was consistent with the theory that the OPG product formed dimers formed as a result of oxidation of the free sulfhydryl group (s).

The putative mature OPG polypeptide contains 23 cysteine residues, of which 18 are believed to be involved. formation of disulfide bridges within the chain that contain four cysteine-enriched domains (FIG. 12A). The five remaining C-terminal cysteine residues are not part of the secondary structure as predicted by homology with other members of the TNFR family. Thus, the putative mature OPG polypeptide contains an overall odd number of cysteine residues and it is theoretically possible that at least one of these

01-1718-97 English ► ··· »« «

The 119 residues are free to form an intermolecular disulfide bond between the two OPG monomers.

Pulse-chase labeling studies have helped elucidate the patterns of OPG kinesis and monomer linkages. CHO cells expressing muOPG [22-401] metabolically labeled with 30 minutes as described above in a medium free of serum and containing ³⁵ S methionine and cysteine. After this period of time, the medium was removed and replaced with complete medium containing unlabeled methionine and cysteine at concentrations approximately 2000-fold greater than the original concentration of radioactive amino acids. 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 12 hours after the addition, the cultures were harvested by removing the conditioned medium and prepared lysates of the conditioned medium and adherent monolayers. The culture medium and cell lysates were purified as described above and subsequently immunologically precipitated using anti-OPG antibodies. To wash the immunological precipitates, they were released by boiling in non-reducing SDS-PAGE buffer and then divided into two halves. To one half was added a reducing agent, β-mercaptoethanol to a final five percent (v / v) concentration, while the other half was maintained in non-reducing conditions. Both sets of immunoprecipitates were analyzed by SDS-PAGE as described above and subsequently processed for autoradiography and exposed to film. The results are shown in Figure 16. Samples of the analyzed SDS-PAGE are shown at the bottom of the figure. After synthesis, the OPG peptide was rapidly processed to a slightly larger polypeptide, which probably represents a modification of N-linked glycosylation. After about 1-2 hours, the level of OPG in the cell decreased sharply and appeared simultaneously in the culture supernatant. This seems to be

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·· 9 ·· '99 • 9 9 9 / · 9 9 9 • • 9 99 · 9 9 9 9 · · 9999 999

This is due to the vector transport of OPG from the cell to the medium over time, consistent with the theory that OPG is a naturally secreted protein. Analysis of the same immunological precipitates under non-reducing conditions reveals a correlation between the formation of OPG dimers and secretion into the conditioned medium (Figure 1.6, upper part). During the first 30 to 60 minutes, OPG monomers were processed in the cell by direct glycosylation followed by dimer formation. Gradually, the volume of OPG monomer was divided into two dimers, which subsequently disappeared from the cell. Approximately 60 minutes after the synthesis was performed, OPG dimers appeared in conditioned medium where they accumulated throughout the duration of the experiment. After this period of time, OPG dimers were formed and subsequently secreted into the culture medium. OPG monomers remained at a low concentration inside the cell during the experiment, and a small amount of these monomers also appeared in the medium. This does not appear to be due to the break of covalent OPG dimers, but rather to the production of a substoichiometric amount of monomers in the cell and their subsequent secretion.

OPG recombinantly generated from transfected CHO cells appears to be predominantly dimeric. In order to determine whether dimerization is a natural process occurring within OPG synthesis, it was examined whether naturally expressed OPG is present in the conditioned cell line medium. Searching RNA tissue and cell lines revealed that the CTLL-2 cell line, the murine cytotoxic T lymphocyte cell line (ATCC Accession Number TIB-214), expresses OPG mRNA. The OPG transcript has been shown to correspond to the cloned and sequenced 2.5 to 3.0 kb RNA identified by the <RTIgt;

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121 in the kidney, and that encodes a secreted molecule. Western analysis of the conditioned media obtained from CTLL-2 cells showed that most, if not all, of the secreted OPG protein was in the form of a dimer (Fig. 17). This finding suggests that OPG dimerization and secretion is not the result of overexpression in the cell line, but that the dimeric form is probably the major form of the product that is generated by the expression cells.

Tissues and serum of normal and transgenic mice were analyzed to determine the nature of the OPG molecule expressed in OPG transgenic mice. Since rat OPG cDNA was expressed under the control of the hepatocyte control element, extracts prepared from the parenchyma of control and transgenic mice under non-reducing conditions were used for analysis (Figure 18). In the extract obtained from transgenic mice, but not in the control mouse, OPG dimers were readily determined along with a substoichiometric amount of monomers. OPG dimers and monomers appear to be identical to recombinant mouse protein expressed in genetically engineered CHO cells. These findings suggest that OPG dimers are in fact a natural form of the gene product and that they are probably key active ingredients. Serum samples obtained from control and transgenic mice were also analyzed by Western analysis. In control mice, most of the OPG protein migrated as a dimer and a small amount of monomer was also analyzed. In addition, a substantial amount of a protein derived from a larger OPG that migrated with a relative molecular weight corresponding to the predicted size of the covalently bound trimer was detected. Thus, recombinant OPG is expressed in

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122

OPG transgenic mice predominantly as a dimer protein and this dimer form may be the basis for the osteopetrotic phenotype in OPG mice. The OPG recombinant protein may also exist in a higher molecular "trimeric form."

To determine whether the five C-terminal cysteine residues of OPG play a role in homodimerization, the murine OPG codons for cysteine residues 195 (C195), C202, C277, C319 and C400 were changed to serine as described above using the QuickChange Site kit as described above. -Directed Mutagenesis Kit (Stratagene, San Diego, CA). The MuOPG gene was subcloned between the Not I and Xba I sites of the pcDNA 3.1 (+) vector (Invitrogen, San Diego, CA). The resulting plasmid pcDNA3.1-muOPG and mutagenic primers were treated in the presence of Pfu deoxynucleotides with polymerase and subsequently amplified in a thermocycler as described above. An aliquot of the reaction mixture was then heat shock transfected into competent E. coli XL1-Blue and finally plated. Plasmid DNA from the transformants was subsequently sequenced to verify mutations.

The following primer pairs were used to change the codon for cysteine residue 195 to the serine of the mouse OPG gene, resulting in muOPG [22-401] C195S protein:

1389-19:

5 '-CAC GCA AAA GTC GGG AAT AGA TGT CAC-3' (SEQ ID NO: 150)

1406-38:

5 '-GTG ACA TCT ATT CCC GAC TTT TGC GTG-3' (SEQ ID NO: 151)

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123

The following primer pairs were used to change the codon for cysteine residue 202 to the serine of the mouse OPG gene, resulting in the formation of the muOPG [22-401] C202S protein:

1389-21:

5 '-CAC CCT GTC AGGA GGC CTT CTT C-3' (SEQ ID NO: 152)

1389-22:

5 '-GAA GAA GGC GTC TTC CGA GGT G-3' (1389-22) (SEQ ID NO: 153)

The following primer pairs were used to change the codon for cysteine residue 277 to the serine of the mouse OPG gene, resulting in muOPG [22-401] C277S protein formation:

1389-23:

5 '-TGA CCT CTC GGA AAG CAG CGT GCA-3' (SEQ ID NO: 154)

1389-24:

5 '-TGC ACG CTG CTT TCC GAG AGG TCA-3' (SEQ ID NO: 155)

The following primer pairs were used to change the codon for cysteine residue 319 to the serine of the mouse OPG gene, resulting in muOPG [22-401] C319S protein:

1389-17:

5 '-CCT AGA AAT CGA GCG AGC AGC TCC-3' (SEQ ID NO: 156)

1389-18:

5 '-CGA TTT CGA GGT CTT TCT CGT TCT C-3' (SEQ ID NO: 157)

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The following primer pairs were used to change the codon for cysteine residue 400 to the serine of the mouse OPG gene, resulting in the formation of muOPG [22-401] C400S protein:

1406-72: /

5 '-CCG TGA AAA TAA GCT CGT TAT AAC TAG GAA TGG-3' (SEQ ID NO: 158)

1406-75:

5 '-CCA TTC CTA GTT ATA ACG AGC TTA TTT TCA CGG-3' (SEQ ID NO: 159)

Each resulting muOPG [22-401] plasmid containing the respective mutation was subsequently transfected into human 293 cells, and the mutant OPG-Fc fusion protein was purified from the conditioned medium as described above. The biological activity of each protein was determined using an in vitro osteoclast formation assay as described in the Example

Conditioned medium from each transfectant was analyzed by non-reducing SDS-PAGE and western blotting with anti-OPG antibodies.

Mutation of any of the five C-terminal cysteine residues results in the production of predominantly (> 90%) monomeric 55 kd OPG molecules. These results suggest that terminal cysteine residues play a role in OPG homodimerization.

C-terminal OPG deletion mutations were constructed into a map (s) of the OPG C-terminal domain (s) that are important for OPG homodimerization. To construct these OPG mutants, PCR amplification was used using primers that introduced immature termination signals to terminate translation into

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C-terminal region of mouse OPG. The 5 'oligonucleotide and the 3' oligonucleotide (containing the termination codon and the XhoI site) were labeled at the MuOPG initiation codon (containing the HindIII restriction site) - to modify the C-terminal portion of the muOPG ending at either threonine residue 200 (CT200), proline 212 (CT212). ), glutamic acid 293 (CT-293) or lilac 355 (CT-355).

The following primers were used for the muOPG construct [22200]:

1091-39:

5 '-CCT CTG AGC TCA AGC TTC CGA GGA CAA CAA TGA ACA AG-3' (SEQ ID NO: 160)

1391-91:

5 '-CCT TCG AGT CAG GTG ACA TCT ATT CCA CAC TTT TGC GTG GC-3' (1391-91) (SEQ ID NO: 161)

The following primers were made to construct muOPG [22-212]:

1091-39:

5 '-CCT CTG AGC-TCA AGC-TTC CGA GGA CCA CAA TGA ACA AG-3' (SEQ ID NO: 162)

1391-90:

5 '-CCT CTC TCG AGT CCA GGA ACA GCA AAC CTG AAG AAG GC -3' (SEQ ID NO: 163)

The following primers were used for the muOPG construct [22-293]:

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126

1091-39:

5 '-CCT CTG AGC TCA AGC TTC CGA GGA CAA CAA TGA ACA AG-3' (SEQ ID NO: 164)

1391-89:

t

5'- CCT CTC TCG AGT CAC TCT GTG

CC-3 '(SEQ ID NO: 165)

The following primers were made to construct muOPG [22-355]:

1091-39:

5 '-CCT CTG AGC-TCA AGC-TTC CGA GGA CCA CAA TGA ACA AG-3' (SEQ ID NO: 166)

1391-88:

5 'CCT CTC TCG AGG CAG GAT GTT TTC AAG TGG TTG AGG GC-3' (SEQ ID NO: 167)

Each resulting muOPG-CT plasmid containing the appropriate treatment was then transfected into human 293 cells of the mutant OPG-Fc fusion protein purified from the conditioned medium described above. The biological activity of each protein was determined in vitro using the osteoclast formation assay described in Example 11. Conditioned media was also analyzed by non-reducing SDS-PAGE and western blotting with anti-OPG antibodies.

Modification of the C-terminal portion of OPG affects the ability of OPG to form homodimers. CT 355 is predominantly monomeric, although some monomers are also formed. CT 293 appears to form the same molar amounts of monomer and dimer and also form high molecular weight aggregates. However, CT 212 and CT 200 are monomeric.

• ·

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Example 10

Purification of OPG

A. Purification of mammalian OPG-Fc fusion proteins ¹ · - '·

Five liters of conditioned media from 293 cells expressing OPG-Fc fusion protein were prepared as follows. The frozen cell sample was thawed in 10 ml of 293S medium (DMEM-high glucose, 1x L-glutamine, 10% heat-inactivated fetal bovine serum (FBS) and 100 gg / ml hygromycin) and the following day fresh sample was provided. After three days, cells were split at 1:10 and 1:20 dilution into two T175 flasks. Two further 1:10 divisions were performed and divided into 200 T175 flasks. Cells were allowed to grow at this point, i.e., five days after thawing, until they reached an almost continuous layer (approximately three days), aspirated to remove serum, and after washing with 25 ml PBS per flask, into each flask. 25 ml of SF medium (DMEM-high glucose, 1x L-glutamine) was added. The cells were maintained at 5% CO 2 for three days, then harvested, centrifuged and filtered through 0.45m cellulose nitrate filters (Corning).

OPG-Fc fusion proteins were purified using a Protein G Sephsarose column (Pharmacia) equilibrated in PBS. The column size varied depending on the volume of the default media. The conditioned medium, prepared as described above, was loaded into a column which was washed with PBS and 100mM glycine pH 2.7 was used to elute the pure protein. Fractions were collected in tubes containing 1M Tris pH 9.2 to neutralize these fractions as quickly as possible. Protein containing fractions were pooled, concentrated either in Amicon Centricon 10 or

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Centriprep 10 and diafiltered into PBS. Pure protein was stored at -80 ° C.

[22-401] -Fc, [22-180] -Fc, [22-194] -Fc, human [22-401] -Fc, and human [22-201] Fc as well as mice were purified by this procedure. [22-185] -Fc.

B. Preparation of anti-OPG antibodies

Three New Zealand white rabbits (starting weight 2,268 g to 3,629 g) were injected subcutaneously with muOPG [22-401] Fc fusion protein. Each rabbit was immunized on the first day with 50 µg of antigen emulsified in an equal volume of Freunds complete adjuvant. Additional booster doses were administered in the same manner on days 14 and 28, with the complete adjuvant being replaced with Freunsd's incomplete adjuvant for these booster doses. Antibody titers were monitored by EIA. After the second booster dose, antisera gave titers with high antibody levels and 25 ml of blood was collected from each animal. Serum was first passed through an affinity column in which OPG-Fc was immobilized. Anti-OPG antibodies were eluted with Pierce Gentle Elution Buffer containing 1% glacial acetic acid. The eluted protein was then dialyzed into PBS and passed through an Fc column to remove antibodies specific for the Fc portion of the OPG fusion protein. Running through fractions containing anti-OPG specific antibodies was dialyzed into PBS.

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C. Purification of mouse OPG [22-401]

Chromatographic affinity of antibodies

Affinity purified anti-OPG antibodies were diafiltered into binding buffer (0.1M sodium carbonate, pH 8.3, 0.5M NaCl) and mixed for two hours at room temperature with CNBr-activated Sepharose beads (Pharmacia). The resin was washed with more binding buffer at room temperature before blocking the unoccupied sites with 1M ethanolamine (pH 8.0) for two hours. The resin was subsequently washed with a low pH solution (0.1M sodium acetate pH 4.0, 0.5M NaCl) followed by a high pH wash solution (0.1M Tris-HCl pH 8.0, 0.5M NaCl). The last flushes were repeated three times. Finally, the resin was equilibrated with PBS before being loaded onto the column. Blind elution was performed using 0.1 M glycine-HCl, pH 2.5, and the column was then re-equilibrated with PBS.

Subsequently, condensed conditioned medium from CHO cells expressing muOPG was applied to the column at low loading rates [22-401]. The column was washed with PBS until the measured UV absorbance at 280 nm returned to baseline. The protein was first eluted from the column with 0.1M glycine HCl (pH 2.5), then equilibrated with PBS and eluted with a second buffer (0.1M CAPS, pH 10.5), 1M NaCl. The two elution samples were separately diafiltered into PBS and sterile filtered before freezing to -20 ° C.

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Conventional chromatography

The conditioned CHO cell medium was concentrated 23 times in a spiral wound Amicon cartridge (S10Y10) and diafiltered to 20 mM Tris pH 8.0. The diafiltered medium was then applied to a Q-Sepharose HP (Pharmacia) column that was equilibrated with 20 mM Tris pH 8.0. The column was then washed until absorbance at 280 nm reached the baseline again. The protein was eluted with twenty column volumes of the elution system consisting of a 0-300 mM NaCl gradient in Tris pH 8.0. OPG protein was detected by Western blotting of fractions eluted from the column.

Fractions containing OPG were pooled and made up to a final concentration of 300 mM NaCl, 0.2 mM DTT. The NiNTA superose (Qiagen) column was equilibrated with 20 mM Tris pH 8.0, 300 mM NaCl, 0.2 mM DTT, and then pooled fractions were introduced. The column was washed with equilibration buffer until baseline absorbance was reached again. Proteins were eluted from the column with a 0-30 mM imidazole elution gradient in equilibration buffer. The remaining proteins were eluted from the column with 1M imidazole. Western blotting was again used to detect OPG containing fractions.

The pooled fractions from the NiNTA column were dialyzed into 10mm potassium phosphate pH 7.0 and 0.2 mM DTT. The dialyzed fraction was then introduced into a ceramic hydroxyapatite column (Bio-Rad), which was then equilibrated with 10 mM phosphate buffer. After the column was washed, the protein was eluted with a volume of 10-100 mM with an elution gradient of potassium phosphate, which was 20 times the column volume. Then

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131 followed by elution with an equal amount of 100-400 mM phosphate gradient.

OPG was detected by staining with SDS-polyacrylamide gels with coomassie blue and western blotting. After pooling, the fractions were diafiltered into PBS and frozen at -80 ° C. The purified protein was in the form of a monomer and remained in this form even after diafiltration into PBS. This monomer is stable when stored frozen or at pH 5 at 4 ° C. However, when stored at 4 ° C in PBS, it appears to form dimers, trimers and tetramers after one week.

D. Purification of human OPG met [22-401] from E. coli

The bacterial cell paste was suspended in 10 mM EDTA at a concentration of 15% (w / v) using a low shear homogenizer at 5 ° C. The cells were subsequently disrupted by double homogenization at 5 ° C and 103.35 MPa. The resulting homogenized sample was centrifuged for one hour at 5 ° C and 5,000 x g. The centrifuged pellets were washed by low shear homogenization into water at the original homogenization volume and then centrifuged as before. The washed pellets were then solubilized for 30 minutes at room temperature in a 15% (w / v) solution (final concentration) of 6 M guanidine HCl, 10 mM dithiothreitol, 10 mM TrisHCl, pH 8.5. This solution was diluted 30 times with 2 M urea containing 50 mM CAPS, pH 10.5, 1 mM reduced glutathione and then stirred at 5 ° C for 72 hours. OPG was purified from this solution, at 25 ° C, by first adjusting the pH value with acetic acid and then chromatographing it through an SP-HP Sepharose column equilibrated with 25 mM sodium acetate, pH 4.5.

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Column elution was performed with twenty column volumes of a 50 mM to 550 mM linear gradient of sodium chloride in the same buffer, at a flow rate of 0.1 column volume / minute. The upper fractions containing only the desired OPG form were collected and stored at 5 ° C or transferred into phosphate buffered saline by buffer, concentrated by ultrafiltration and then stored at 5 ° C. This material was analyzed by reverse phase HPLC, SDS-PAGE, a limulus amebocytlyzate assay to verify the presence of endotoxin and N-terminal sequencing. In addition, other techniques may be used to determine the folded nature of the protein, such as mass spectrometry, pH / temperature stability assay, fluorescence assay, circular dichroism, differential scanning calorimetry, and protease profiling assays.

Example 11

Biological activity of recombinant OPG

Histological and histomorphometric results suggest that hepatic overexpression of OPG in transgenic mice significantly reduced the number of osteoclasts, causing a substantial increase in bone tissue (see, for example, 4). To better understand the potential mechanism (s) of this in vivo effect, various forms of recombinant OPG were analyzed in an in vitro osteoclast formation model (osteoclast formation assay). This culture system, originally designed by Udagawa (Udagawa et al. Endocrinology 125, 1805-1813 (1989), Proc. Natl. Acad, Sci. USA 87, 7260-7264 (1990)), uses a combination of bone marrow cells and cells from bone marrow supportive cell lines. Description of the modification of this cultivation system used in •

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133 of these studies have already been published publicly (Lacey et al. Endocrinology 136, 2367-2376 (1995)). In this method, bone marrow cells obtained from the femoral and tibial washes of mice were cultured overnight in culture medium (alpha MEM with 10% heat-inactivated fetal bovine serum) supplemented with 500 U / ml CSF-1 (colony stimulating factor) 1, also referred to as M-CSF), i.e., a hematopoietic growth factor specific for cells of the monocyte-macrophage family line. After incubation, the harvested cells, nonadherent, and subjected to gradient purification, and then re-cultured with cells from the cell line ST2 bone marrow (1 x 10 ^e non-adherent cells: 1 x 10 ⁵ ST2 cells / ml media). This medium is enriched with dexamethasone (100 nM) and the biologically active metabolite of vitamin D3, known as 1.25 dihydroxyvitamin D3 (1.25 (OH) 2 D3, 10 nM). To improve osteoclast imaging, prostaglandin E2 (250 nM) was added to some cultures. The co-culture period generally ranges from 8 to 10 days, and fresh medium with all additional ingredients is added every 3 to 4 days. At various intervals, cultures were used to verify the presence of tartrate resistant acid phosphatase (TRAP) using either a histochemical dye (Sigma Kit # 387A, Sigma, St. Louis, MO) or using a TRAP solution assay. The TRAP histochemical method allows phenotypic identification of osteokiasts, which are multinucleated (> 3 nuclei) cells, which are also TRAP +. The essence of the solution test is the cleavage of cultures containing osteoclasts in citrate buffer (100 mM, pH 5.0) containing 0.1% Triton X-100. The activity of tartrate resistant acid phosphatase was then determined by converting p-nitrophenyl phosphate (20 nM) to p-nitrophenol in the presence of 80 mM sodium tartrate,

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134 which occurred during a three to five minute incubation at room temperature. The reaction was terminated by adding NaOH to a final concentration of 0.5 M. The optical density was measured at 405 nm and the results were plotted. ......, ......

Previous studies (Udagawa et al., Ibid.) Conducted using an osteoclast formation assay have shown that cells also express ¹²⁵ I-calcitonin receptors (autoradiography) and induce bone surface pits and, when combined with TRAP positivity, confirm that multinucleated cells have an osteoclast phenotype. Further evidence of support for the osteoclast phenotype of multinucleated cells obtained in the in vitro osteoclast formation assay is that cells express α 1 and β 3 integrins by immunocytochemistry and calcitonin receptor and TRAP mRNA by in situ hybridization (ISH).

The HuOPG [22-401] -Fc fusion was purified from CHO cells of conditioned medium and subsequently used in the osteoclast formation assay. At 100 ng / ml huOPG [22-401] -Fc, osteoclast formation was inhibited substantially by 100% (Fig. 19A). TRAP levels, as measured in lysed cultures in microtiter wells, were also inhibited in the presence of OPG with an ID _{50 of} approximately 3 ng / ml (Fig. 20). The level of TRAP activity in the lysates appeared to correlate with the relative number of osteoclasts detected by TRAP cytochemistry (compare Figures 19A to 19G and 20). Purified human IgG1 and TNFbp were also analyzed in this model and found to have no inhibitory or stimulatory effects, suggesting that the inhibitory effects of huOPG [22-401] -Fc are effects caused by the OPG portion of the fusion protein. Other forms of human and murine molecules were analyzed in the same way, and the cumulative results obtained are summarized in Table 1 below.

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Table 1

Effects of various OPG forms on in vitro osteoclast formation

OPG construct Relative bioactivity in vitro

muOPG [22-401] -Fc +++ muOPG [22-194] -Fc +++ muOPG [22-185] -Fc ++ muOPG [22-180] -Fc - muOPG [22-401] +++ muOPG [22-401] C195 +++ muOPG [22-401] C202 + muOPG [22-401] C277 - muOPG [22-401] C319 + muOPG [22-401] C400 + muOPG [22-185] - muOPG [22-194] ++ muOPG [22-200] ++ muOPG [22-212] - muOPG [22-293] +++ muOPG [22-355] +++ huOPG [22-401] -Fc +++ huOPG [22-201] -Fc +++ huOPG [22-4011-Fc P26A +++ - huOPG [22-401] -Fc Y28F +++ ’ huOPG [22-401] +++ huOPG [27-401] -Fc ++ huOPG [29-401] -Fc ++ huOPG [32-401] -Fc +/-

+++, ED ₅₀ = 0.4-2 ng / ml ++, ED ₅₀ = 2-10 ng / ml + / ED ₅₀ = 10-100 ng / ml ED ₅₀ > 100 ng / ml

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The above summary data suggests that murine and human OPG amino acid sequences 22-401 are fully active in vitro both in the form fused to the Fc domain and in the non-fused form. They inhibit in a dose-dependent manner and show half to maximal activity at doses of 2 to 10 ng / ml. Modification of the mouse C-terminus at threonine residue 180 inactivates the molecule, while modifications to cysteine 185 and further retain full activity. The cysteine residue at position 185 is predetermined for the formation of SS3 binding in the domain region of OPG. Removal of this residue in other TNFR-related proteins has previously shown inhibition of biological activity (Yan et al., J. Biol. Chem. 226, 12099-12104 (1994)). Our finding that muOPG [22-180] -Fc is inactive while muOPG [22-185] -Fc is active is consistent with earlier findings. Accordingly, amino acid residues 22-185 define the region of OPG activity.

The results of osteoclast formation assays also show that, like transgenically expressed OPG, recombinant OPG protein suppresses osteoclast formation. Experiments analyzing the discovery of TRAP + cells, β3 + cells, F480 + cells in cultures continuously exposed to OPG have shown that OPG blocks the appearance of TRAP + and β3 + cells but not F480 + cells. On the other hand, TRAP + and β3 + cells started appearing four days after the culture appeared in control cultures. Only F480 + cells can be found in OPG-treated cultures and appear to be present in quantitatively equal numbers as in control cultures. Thus, the in vitro OPG mechanism appears to be involved in blocking osteoclast differentiation in the step involving the formation of mohocyto-macrophages, but prior to

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137 cells expressing either TRAP or β3 integrins. Taken together, these findings suggest that OPG does not interfere with the general growth and differentiation of monocyte-macrophage bone marrow precursors, but rather concludes that OPG-specifically blocks the selective differentiation of osteoclasts from monocytomacrophage precursors.

Variation of the in vitro culture method was used to more accurately time the time at which OPG appears to be an inhibitor during osteoclast differentiation. This variation, described by Lacey et al., Below, utilizes bone marrow macrophages as osteoclast precursors. Osteoclast precursors are obtained by harvesting non-adherent bone marrow cells after overnight incubation in CSF-1 / M-CSF, and culturing these cells for an additional four days with 1,000-2,000 U / ml CSF-1. After four days of cultivation, i. growth phase, non-adherent cells were removed. Adherent cells, which are bone marrow macrophages, can then be subjected to a maximum of two days of various treatments in the presence of 1000 to 2000 U / ml CSF-1. This two-day period is referred to as the mean differentiation period. The cell layers were then allowed to grow again and ST-2 cells (1 x 10 ⁵ cells / ml), dexamethasone (100 nM) and 1.25 (OH) 2 D 3 (10 nM) were added for at least eight days. This time period is referred to as the final differentiation period. Analytical reagents may also be administered during this final differentiation period. Phenotypic markers of osteoclast differentiation are also obtained during the final differentiation period (Lacey et al., Ibid.).

The effect of huOPG [22-401] -Fc (100 ng / ml) on osteoclast formation was analyzed in this model by adding this fusion peptide during either the intermediate or final

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138, or alternatively during both of these periods. Both the TRAP cytochemical and TRAP solution tests were performed. The results of the TRAP solution assay are shown in Figure 21. HuOPG [22-401] -Fc · inhibits the formation of TRAP activity when added to both the intermediate and final differentiation phases, or both. If added to the middle phase and subsequently rinsed from the cultures, then huOPG [22-401] -Fc did not block the formation of TRAP activity in the culture lysates. Cytochemical results were parallel to the data obtained with the solution test. The results of all these tests indicate that its presence during the final differentiation period is important to exert all the suppressive effects of huOPG [22-401] -Fc on osteoclast formation.

B. In vivo IL1-α and IL1-β immunological experiments

IL1 enhances bone resorption, both systemically and locally when injected subcutaneously through mouse calva (Boyce et al., Endocrinology 125, 1142-1150 (1989)).

Systemic effects can be derived from the degree of hypercalcemia and local effects can be determined histologically by determining the relative value of the osteoclast-mediated response. The aim of these experiments was to determine. Whether recombinant muOPG [22-401] -Fc can modify the local and / or systemic effects of IL1 when injected subcutaneously across the calva region, as in the case of TLI.

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139

IL1-β experiment

Male mice (ICR Swiss white), four weeks old, were divided into the following groups for treatment purposes (five mice per group). Control group: IL1 treated animals (mice received one injection ^- 2.5 gg III-β per day); low muOPG [22-401] -Fc treated animals group (mice received three injections of 1 gg muOPG [22-401] -Fc daily); low dose muOPG [22-401] Fc and IL1-β group treated mice; a group of animals treated with a high dose of muOPG [22-401] -Fc (mice received three injections of 10 gg of muOPG [22-401] -Fc daily); and a group of mice treated with a high dose of muOPG [22-401] -Fc and IL1-β. All mice received the same total number of injections of either active factor or vehicle (0.1% bovine serum albumin in phosphate buffered saline). Animals from all groups were sacrificed the day after the last injection. The weight and level of ionized calcium in the blood was determined before the first injection, four hours after the second injection and 24 hours after the third IL1 injection, and the last measurement was made immediately before the animals were sacrificed. After the animals were sacrificed, the calva was removed from the animals and further processed for paraffin separation.

IL1-α experiment

Male mice (ICR Swiss white), four weeks old, were divided into the following groups for treatment (five mice per group). Control group: IL1-alpha treated animals (mice received a single injection of 5 gg IL1-a

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140 per day); a group of low-dose muOPG [22-401] -Fc animals (mice received a single injection of 10 gg muOPG [22401] -Fc daily); a group of mice treated with low dose muOPG [22-401] -Fc and IL1-d (dosing see above); a group of animals treated with a high dose of muOPG [22-401] -Fc (mice received three injections of 10 gg of muOPG [22-401] -Fc daily); and a group of mice treated with a high dose of muOPG [22-401] -Fc and IL1-α. All mice received the same total number of injections of either active factor or vehicle. Animals from all groups were sacrificed the day after the last injection. The level of ionized calcium in the blood was determined before the first injection, four hours after the second injection and 24 hours after the third IL1 injection, and the last measurement was made immediately before the animals were sacrificed. The weight of the animals was determined before the first injection, four hours after the second injection and 24 hours after the third IL1 injection, and the last measurement was made immediately before the animals were sacrificed. After the animals were sacrificed, the calva was removed from the animals and further processed for paraffin separation.

Histological methods

......... Calvary bone samples were fixed with zinc formalin, decalcified in formic acid, dehydrated with ethanol and paraffin-treated. Sections (5 μιη thick) were guided through the calva beside the lambda seam and stained with either hematoxylin and eosin or reacted for tartrate-resistant acid phosphatase activity (Sigma Kit # 387A) and stained with hematoxylin antibody. Bone resorption was evaluated in IL1-α treated mice by histomorphometric methods

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141 use of oste value (Osteimetrics, Atlanta, GA) by observing histological features on a digitizer plate using a microscope to which the camera is attached to the eyepiece portion with the appropriate extension. The number of osteoclasts, osteoclast-coated surfaces and eroded surfaces were determined in the main marrow spaces of the calvary bone. Injected and uninjected sides of the calvum were measured separately.

Results

IL1-α and IL1-β produced hypercalcemia at the doses used, particularly on the second day, probably due to systemic induction of increased bone resorption. The hypercalcemic response was blocked by muOPG [22401] -Fc in IL1-beta treated mice and was significantly attenuated in IL1-alpha treated mice. This effect was most evident on the second day (Figure 22A to 22B).

Histological analysis of the calvus of IL1-α and IL1-β-treated mice shows that IL1-alone treatment produces a substantial increase in bone resorption indications including: number of osteoclasts, osteoclast-lined surface and eroded surface (surfaces that are indented by osteoclasts (Fig. 23B, In response to IL1-α or IL1-β, the increase in bone resorption on the injected and uninjected sides of the calva was essentially the same Muopg [22-401] -Fc injections reduce bone resorption in both IL1-α and IL1- treated mice. β in mice receiving vehicle alone, but this reduction was only observed on the sides of the calf injected with muopg [22-401] -Fc.

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The most likely explanation for these observations is that muOPG [22-401] -Fc inhibited bone resorption. This conclusion is based on the observed reduction in the total number of osteoclasts and the percentage of bone surface at which bone resorption can occur in the area of the calva adjacent to muOPG [22-401] -Fc injection sites. The action of muOPG [22-401] -Fc appears to be rather local, as histology has shown, but the fact that muOPG [22-401] -Fc also inhibits IL1-induced hypercalcemia suggests that muOPG [22-401] -Fc it has a much milder influence on the systemic concept of bone resorption.

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C. Systemic effects of muOPG [22-401] -Fc in growing mice

Three to four week old male BDF1 mice weighing 9.2-15.7 g were divided into groups of ten mice. Then, mice were injected subcutaneously with saline or muOPG [22-401] -Fc 2.5mg / kg of misery for fourteen days (5mg / kg / day). Mice were radiographed before treatment and on days 7 and 14 after treatment. 24 hours after the last injection, mice were sacrificed. The right femur was removed, fixed in zinc formalin, decalcified in formic acid, and encapsulated in paraffin. Sections were guided through the central area of the distal femoral metaphysis and the central portion of the femur. Bone density was determined histomorphometrically in six contiguous areas, ranging from the metaphyseal boundary plate of the growth plate through primary and secondary spongiosis to femoral diaphysis (midbone). All areas were 0.5 x 0.5 mm ² .

Radiographic changes

After seven days of treatment, spongiosis associated with growth plates of OPG-treated mice showed a zone of increased bone density, compared to the bone density observed in this area in control mice. The effects were particularly pronounced in the distal femoral and proximal tibial metaphase (Fig. 2). 24A to 24B). However, bands of increased density also appeared in vertebral bodies, at the iliac edge and distal tibia. After 14 days, the areas of opacity expanded further into the central part of the femoral and tibial bones, but in terms of the intensity of radio opacity, it decreased. After completion of

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In addition, no differences in the length of the middle parts of the femoral and tibial bones or changes in the length of these bones during the experiment were observed, suggesting that OPG does not affect bone growth. .

Histological changes

Distal femoral metaphysis exhibits increased bone density in regions 1.1 to 2.65 mm distant from the growth plate (Figs. 25 and 26A to 26B). This is the area in the body of mice from which bone is rapidly removed by osteoclastymediated bone resorption. In these fast-growing young mice, the increase in bone density in this region observed during OPG treatment corresponds to inhibition of bone resorption.

D. Effects of osteoprotegerin on ovarianectomy-induced bone loss in rats

Twelve-week-old female Fisher rats were subjected to ovariectomy (OVX) or the same ovariectomized surgery and performed two X-ray absorption photometry (DEXA) measurements to determine bone density in distal femoral metaphysis. After a three-day convalescence period, animals were injected daily for fourteen days according to the following schemes: ten shadow-operated animals received vehicle (phosphate buffered saline); ten OVX animals received vehicle (phosphate buffered vehicle); six animals received OPG-Fc 5 mg / kg SC;

··

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146 six OVX animals received pamidronate (PAM) 5 mg / kg SC; Six OVX animals received estrogen (ESTR) 40 μς / kg SC. After seven to fourteen days of treatment the animals was measured using DEXA bone density mass ^7. Two days after the last injection, the animals were sacrificed and the right tibial and femur bones were harvested for histological evaluation.

DEXA bone density measurements showed a tendency to bone loss due to ovariectomy that was blocked by OPG-Fc. These effects are essentially the same as those of known antiresorptive agents, estrogen and pamidronate (Fig. 27). Histomorphometric analysis confirmed the observation that in OPG-Fc treatment, bone mass production was significantly greater in OVX rats than bone production that was observed in untreated OVX rats (Fig. 28). These results confirm OPG activity in bone loss associated with endogenous estrogen deficiency after ovariectomy.

Summary in vivo

The effects of recombinant OPG in vivo are a parallel to the changes observed in OPG transgenic mice. The reduction in the number of osteoclasts observed in OPG transgenic mice is reproduced by injecting recombinant OPG locally across the calvum of both normal and IL1-α or IL1-β treated mice. OPG transgenic mice developed an osteopetrotic phenotype with progressive bone marrow filling and non-remodeled cartilage extending from the growth plates from day one after birth. In normal three-week (growing) mice, OPG treatment also led to ·· «·

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147 bone retention and non-remodeled cartilage in areas of endochondral bone formation. This effect was monitored by radiography and confirmed histologically. Thus, recombinant OPG induces phenotypic changes in normal animals similar to those seen in transgenic animals, and these changes correspond to OPG-induced inhibition of bone resorption. Based on the results of in vitro tests for osteoclast formation, it was found that much of the inhibition occurs due to impaired osteoclast formation. Consistent with this hypothesis, OPG blocks ovariectomy-induced osteoporosis in rats. It is known that bone loss in this model is mediated by activated osteoclasts, suggesting a role for OPG in the treatment of primary osteoporosis.

Example 12

Pegylation of OPG derivatives

Preparation of N-terminal PEG-OPG conjugates by reductive alkylation

HuOPG met [22-194] P25A formed a buffer, which was transferred to 25 to 50 mM NaOAc, pH 4.5 to 4.8 and concentrated to 2-5 mg / ml. This solution was used to perform OPG reductive alkylation ^with monofunctional PEG aldehydes at 5 to 7 ° C. PEG monofunctional aldehydes, linear or branched, ΜΗ = 1 to 57 kDa (available from Shearwater Polymers), were added to the OPG solution as solids in amounts of 2-4 moles of PEG aldehyde per mole of OPG. After dissolution of the polymer in the protein solution, sodium cyanoborohydride was added in an amount to give a final concentration of 15-20 mM in the reaction mixture of 1 to 1.6 M

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148 freshly prepared stock solution in cold DI water. Reaction progress and extent of OPG pegylation was monitored by size exclusion HPLC on a G3000SW _XL column (Toso Haas) eluted with 100 mM NaPO ₄ , 0.5 M NaCl, 10% ethanol, pH-6.9. The reaction was generally allowed to proceed for 16 to 18 hours, then diluted 6 to 8 times and the pH was lowered to 3.5 to 4. The reaction mixture was fractionated by ion exchange chromatography (HP SP HiLoad 16/10, Pharmacia) eluting with 20 min. mM NaOAc pH 4 with a linear gradient to 0.75M NaCl over 25 column volumes and at a flow rate of 30 cm / hr. Mono-, di- or poly-pegylated OPG fractions were collected and characterized by SEC HPLC and SDS-PAGE. N-terminal sequencing revealed that the monoPEG-OPG conjugate, in most cases the major reaction product, was 98% N-terminal PEG-modified OPG.

This procedure was generally used to prepare the following N-terminal PEG-OPG conjugates (in which OPG HuOPG met [22-194] P25A: 5 kD monoPEG, 10 kD mono-PEG, 12 kD monoPEG, 20 kD monoPEG, 20 kD mono-PEG 25 kD monoPEG, 31 kD monoPEG, 57 kD monoPEG, 12 kD diPEG, 25 kD diPEG, 31 kD diPEG, 57 kD diPEG, 25 kD triPEG.

Preparation of PEG-OPG conjugates by acylation

HuOPG met [22-194] P25A formed a buffer, which was transferred to 50 mM BICINE buffer, pH 8 and concentrated to 2-3 mg / ml. This solution was used to perform OPG reductive acylation with monofunctional PEG N-hydroxysuccinimide esters performed at room temperature. PEG N-hydroxysuccinimidyl esters, linear or branched, ΜΗ = 1 to 57 kDa (available)

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149 from Shearwater Polymers) were added to the OPG solution as solids in amounts of 4 to 8 moles of PEG N-hydroxysuccinimidyl ester per mole of OPG. Reaction progress and extent of OPG pegylation was monitored by size exclusion HPLC on a G3000SW _xl column (Toso Haas) eluted with 100 mM NaPO ₄ , 0.5 M NaCl, 10% ethanol, pH 6.9. The reaction was typically allowed to proceed for 1 hour, after which the reaction mixture was diluted 6-8 times and the pH was lowered to 3.5-4. The reaction mixture was fractionated by ion exchange chromatography (HP SP HiLoad 16/10, Pharmacia) eluting with 20 mM NaOAc pH 4 with a linear gradient to 0.75M NaCl over 25 column volumes at a flow rate of 30 cm / hr. Mono-, di- or poly-pegylated OPG fractions were collected and characterized by SEC HPLC and SDS-PAGE.

This procedure was generally used to prepare the following PEG-OPG conjugates: 5 kD polyPEG, 20 kD polyPEG, 40 kD branched PEG, 50 kD polyPEG.

Preparation of dimeric PEG-OPG

HuOPG met [22-194] P25A was prepared by thiolation at 1-3 mg / ml in phosphate buffer at near neutral pH. To S-acetyl mercaptosuccinic anhydride (AMSA) was added, with stirring, at 4 ° C, over a period of two hours, to a 3- to 7-fold molar excess while maintaining the pH at 7.0. The monomethylated OPG was separated from unmodified and polythiolated OPG by ion exchange chromatography, and the protected thiol was deprotected by hydroxylamine treatment. After deprotection, hydroxylamine was removed by gel filtration and the resulting monothiolated OPG was subjected to a variety of thiol treatments.

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• 9 · 9 · 9 · 9 · 9 · 9

99 99

150 specific cross-linking chemical reactions. To form the disulfide-bound dimer, the thiolated OPG at > 1mg / ml was allowed to undergo air oxidation by dialysis in a slightly basic phosphate buffer. The covalent thioether OPG dimer was prepared by introducing a bismaleinimide crosslinking agent, N, N-bis (3-maleimido propianyl) -2-hydroxy 1,3-propane, with thiolated OPG at> 1mg / ml at a 0.6 fold molar ratio of crosslinking agent to OPG in phosphate buffer at pH 6.5. Similarly, dumbbells of PEG are obtained by reacting substoichiometric amounts of bis-maleimide PEG crosslinking agents with thiolated OPG at 1 mg / ml in phosphate buffer at 6.5. Any of the above dimer conjugates could be further purified by either ion exchange or size exclusion chromatography.

Dimeric PEG-OPG conjugates (in which OPG is HuOPG met [22-194] P25A), prepared using the procedures described above, include disulfide-linked OPG dimer, covalent thioether OPG dimer with an amine-type aliphatic crosslinking agent, 3.4 kD and 8 kD dumbbells and monobells.

PEG-OPG conjugates were tested for in vitro activity using the osteoclast maturation assay described in Example 11A and ‘in vivo activity by measuring bone density increase after injection into the body of mice performed as described in Example 11C. The in vivo activity is summarized in Table 3 below.

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151

Table 3

In vivo biological activity of pegylated OPG

OPG construct

P25A P25A 5k PEG P25A 20k PEG P25A 31k PEG P25A 57k PEG P25A 12k PEG P25A 20k branched

muOPG met [22-194] muOPG met [22-194] muOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194] huOPG met [22-194]

5k PEG 20k PEG

P25A 8k PEG dimer P25A disulfide crosslink

Increasing the density of the tibial bone +

+ +

+ +

+ +

+ +

+ * * *

In conclusion, it should be noted that the above examples of embodiments of the invention are to be regarded as preferred embodiments, which are illustrative only and not in any way limit the scope of the invention, which is clearly defined by the appended claims.

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SEQUENCE PROTOCOL (1) data for SEQ ID NO: 1:

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AAAGGAAGGA AAAAAGCGGC CGCTACANNN NNNNNT (1) data for SEQ ID NO: 2:

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(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TCGACCCACG CGTCCG (1) data for SEQ ID NO: 3: - = ..........

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(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

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GGGTGCGCAG GC (1) data for SEQ ID NO: 4:

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TGTAAAACGA CGGCCAGT 18 (1) data for SEQ ID NO: 5:

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CAGGAAACAG CTATGACC 18 (1) data for SEQ ID NO: 6:

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CAATTAACCC TCACTAAAGG

data for SEQ ID NO: 7: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 23 basic pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GCATTATGAC CCAGAAACCG GAC 23 (1) data for SEQ ID NO: 8:

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AGGTAGCGCC CTTCCTCACA TTC____ 23 (1) data for SEQ ID NO: 9:

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GACTAGTCCC ACAATGAACA AGTGGCTGTG 30 (1) data for SEQ ID NO: 10:

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ATTAGAATGC GGCCGCTAAA CTATGAAACA GCCCAGTGAC CATTC 45 (1) data for SEQ ID NO: 11:

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GCCTCTAGAA AGAGCTGGGA C '.....,. . ..... - ₂₁ (1) data of SEQ ID NO: 12:

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CGCCGTGTTC CATTTATGAG C ——. (1) data relating to SEQ ID NO: 13:

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ATCAAAGGCA GGGCATACTT CCTG 24 (1) data for SEQ ID NO: 14:

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GTTGCACTCC TGTTTCACGG TCTG 24 (1) data for SEQ ID NO: 15:

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(Ii) MOLECULAR TYPE: cDNA

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CAAGACACCT TGAAGGGCCT GATG (1) data for SEQ ID NO: 16:

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TAACTTTTAC AGAAGAGCAT CAGC 24 (1) data for SEQ ID NO: 17:

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AGCGCGGCCG CATGAACAAG TGGCTGTGCT GCG 33 (1) data for SEQ ID NO: 18:

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AGCTCTAGAG AAACAGCCCA GTGACCATTC C = .......

(1) Data on SEQ ID NO: 19:

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GTGAAGCTGT GCAAGAACCT GATG 24 (1) data for SEQ ID NO: 20:

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ATCAAAGGCA GGGCATACTT CCTG (1) data for SEQ ID NO: 21:

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CAGATCCTGA AGCTGCTCAG TTTG '- * - 24 (1) data for SEQ ID NO: 22:

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AGCGCGGCCG CGGGGACCAC AATGAACAAG TTG 33 (1) data of SEQ ID NO: 23:

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AGCTCTAGAA TTGTGAGGAA ACAGCTCAAT GGC 33 (1) data for SEQ ID NO: 24:

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ATAGCGGCCG CTGAGCCCAA ATCTTGTGAC AAAACTCAC - 39 (I) data for SEQ ID NO: 25:

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TCTAGAGTCG ACTTATCATT TACCCGGAGA CAGGGAGAGG CTCTT 45 (1) data for SEQ ID NO: 26:

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CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (1) data for SEQ ID NO: 27:

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44 «··· 44 4 4 · 4 4 · • · · · · · · · · 01-1718-97 Če e 4 4 4 <4 4,444 4 4 4 · · · 4 ·

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CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (1) data for SEQ ID NO: 29:

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TCCGTAAGAA ACAGCCCAGT GACC 24 (1) data for SEQ ID NO: 30:

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CCTCTGCGGC CGCTGTTGCA TTTCCTTTCT G - - ·· - ......- 3.1 (1) Data on SEQ ID NO: 31:

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(A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Glu Thr Leu Pro Lys Tyr Leu His Tyr Asp Glu Thr Gly His 1 5 10 15

Gin Leu Leu (1) data for SEQ ID NO: 32:

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TCCCTTGCCC TGACCACTCT T 21 (1) data for SEQ ID NO: 33:

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CCTCTGCGGC CGCACACACG TTGTCATGTG TTGC 34 (1) data for SEQ ID NO: 34:

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TCCCTTGCCC TGACCACTCT T 21 (1) data for SEQ ID NO: 35:

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CCTCTGCGGC CGCCTTTTGC GTGGCTTCTC TGTT 34 (1) SEQ ID NO: 36:

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CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (1) data for SEQ ID NO: 37:

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CCTCTGCGGC CGCTAAGCAG CTTATTTTTTA CTGAATGG 38 (1) data for SEQ ID NO: 38:

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CCTCTGAGCT CAAGCTTGGT TTCCGGGGAC CACAATG 37 (1) Data on SEQ ID NO: 39:

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CCTCTGCGGC CGCCAGGGTA ACATCTATTC CAC 33 (1) data for SEQ ID NO: 40:

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CCGAAGCTTC CACCATGAAC AAGTGGCTGT GCTGC 35 (1) data for SEQ ID NO: 41:

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CCTCTGTCGA CTATTATAAG CAGCTTATTT TCACGGATTG 40 (1) data for SEQ ID NO: 42:

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TCCCTTGCCC TGACCACTCT T 21 (1) data for SEQ ID NO: 43:

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CCTCTGTCGA CTTAACACAC GTTGTCATGT GTTGC 35 (1) data for SEQ ID NO: 44:

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TCCCTTGCCC TGACCACTCT T 21 (1) data for SEQ ID NO: 45:

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CCTCTGTCGA CTTACTTTTG CGTGGCTTCT CTGTT (1) data for SEQ ID NO: 46:

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GTGAAGAGCG TGÁAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT 120 TCGGGCTTTC TTCTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTCGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA AAACAAATCT AGAATCAAAT CGATTAATCG ACTATAACAA ÁCCAŤTTTCT 300 TGCGTAAACC TGTACGATCC TACAGGTACT TATGTTAAAC AATTGTATTT CAAGCGATAT 360 AATAGTGTGA CAAAAATCCA ATTTATTAGA ATCAAATGTC AATCTATTAC CGTTTTAATG 420 ATATATAACA CGCAAAACTT GCGACAAACA ATAGGTAAGG ATAAAGAGAT GGGTATGAAA 480

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168

GACATAAATG CCGACGACAC TTACAGAATA ATTAATAAAA TTAAAGCCTG TAGAAGCAAT 540 AATGATATTA ATCAATGCTT ATCTGATATG ACTAAAATGG TACATTGTGA ATATTATTTA 600 CTCGCGATCA TTTATCCTCA TTCTATGGTT AAATCTGATA TTTCAATTCT GGATAATTAC 660 CCTAAAAAAT GGAGGCAATA TTATGATGAC GCTAATTTAA TAAAATATGA TCCTATAGTA 720. GATTATTCTA ACTCCAATCA TTCACCGATT AATTGGAATA TATTTGAAAA CAATGCTGTA 780 AATAAAAAAT CTCCAAATGT AATTAAAGAA GCGAAATCAT CAGGTCTTAT CACTGGGTTT 840 AGTTTCCCTA TTCATACTGC TAATAATGGC TTCGGAATGC TTAGTTTTGC ACATTCAGAG 900 AAAGACAACT ATATAGATAG TTTATTTTTA CATGCGTGTA TGAACATACC ATTAATTGTT 960 CCTTCTCTAG TTGATAATTA TCGAAAAATA AATATAGCAA ATAATAAATC AAACAACGAT 1020 TTAACCAAAA GAGAAAAAGA ATGTTTAGCG TGGGCATGCG AAGGAAAAAG CTCTTGGGAT 1080 ATTTCAAAAA TATTAGGCTG TAGTAAGCGC ACGGTCACTT TCCATTTAAC CAATGCGCAA 1140 ATGAAACTCA ATACAACAAA CCGCTGCCAA AGTATTTCTA AAGCAATTTT AACAGGAGCA 1200 ATTGATTGCC CATACTTTAA AAGTTAAGTA CGACGTCCAT ATTTGAATGT ATTTAGAAAA 1260 ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG TCAGGATGGC CTTCTGCTTA 1320 ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC ACCCTCCGGG CCGTTGCTTC 1380 GCAACGTTCA AATCCGCTCC CGGCGGATTT GTCCTACTCA GGAGAGCGTT CACCGACAAA 1440 CAACAGATAA AACGAAAGGC CCAGTCTTTC GACTGAGCCT TTCGTTTTAT TTGATGCCTG 1500 GCAGTTCCCT ACTCTCGCAT GGGGAGACCA TGCATAC 1537 (1) Data on SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA

01-1718-97 Če

169 (1, SEQ ID NO: 48 data):

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs ^J (B) TYPE: nucleic acid -. ,, (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55 (1) data for SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

AND

CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (1) data for SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1546 basic '*' pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50 :

GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA

01-1718-97 Če

170

CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGCCATC 240 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 300 AAATATGGAC GTCGTACTTA ACTTTTAAAG TATGGGCAAT CAATTGCTCC TGTTAAAATT 360 GCTTTAGAAA TACTTTGGCA GCGGTTTGTT GTATTGAGTT TCATTTGCGC ATTGGTTAAA 420 TGGAAAGTGA CCGTGCGCTT ACTACAGCCT aatatttttg AAATATCCCA AGAGCTTTTT 480 CCTTCGCATG CCCACGCTAA ACATTCTTTT TCTCTTTTGG TTAAATCGTT GTTTGATTTA 540 TTATTTGCTA TATTTATTTT TCGATAATTA TCAACTAGAG AAGGAACAAT TAATGGTATG 600 TTCATACACG CATGTAAAAA TAAACTATCT ATATAGTTGT CTTTCTCTGA ATGTGCAAAA 660 CTAAGCATTC CGAAGCCATT ATTAGCAGTA TGAATAGGGA AACTAAACCC AGTGATAAGA 720 CCTGATGATT TCGCTTCTTT AATTACATTT GGAGATTTTT TATTTACAGC ATTGTTTTCA 780 AATATATTCC AATTAATCGG TGAATGATTG GAGTTAGAAT AATCTACTAT AGGATCATAT 840 TTTATTAAAT TAGCGTCATC ATAATATTGC CTCCATTTTT TAGGGTAATT ATCCAGAATT 900 GAAATATCAG ATTTAACCAT AGAATGAGGA TAAATGATCG CGAGTAAATA ATATTCACAA 960 TGTACCATTT TAGTCATATC AGATAAGCAT TGATTAATAT CATTATTGCT TCTACAGGCT 1020 TTAATTTTAT TAATTATTCT GTAAGTGTCG TCGGCATTTA TGTCTTTCAT ACCCATCTCT 1080 TTATCCTTAC CTATTGTTTG TCGCAAGTTT TGCGTGTTAT ATATCATTAA AACGGTAATA 1140 GATTGACATT TGATTCTAAT AAATTGGATT TTTGTCACAC TATTATATCG CTTGAAATAC 1200 AATTGTTTAA CATAAGTACC TGTAGGATCG TACAGGTTTA CGCAAGAAAA TGGTTTGTTA 1260 TAGTCGATTA ATCGATTTGA TTCTAGATTT GTTTTAACTA ATTAAAGGAG GAATAACATA 1320 TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT GCAGGGTACC ATGGAAGCTT 1380 ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC CCGAAAGGAA GCTGAGTTGG 1440 CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG GGCCTCTAAA CGGGTCTTGA 1500 GGGGTTTTTT GCTGAAAGGA GGAACCGCTC TTCACGCTCT TCACGC 1546

(1) Data on SEQ ID NO: 51:

• ·

01-1718-97 Ce

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

TATGAAACAT CATCACCATC ACCATCATGC TAGCGTTAAC GCGTTGG 47 (1) Data on SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

AATTCCAACG CGTTAACGCT AGCATGATGG TGATGGTGAT GATGTTTCA 49 (1) data for SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 141 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

CTAATTCCGC TCTCACCTAC CAAACAATGC CCCCCTGCAA AAAATAAATT CATATAAAAA

01-1718-97 Če

172

ACATACAGAT AACCATCTGC GGTGATAAAT TATCTCTGGC GGTGTTGACA TAAATACCAC 120

TGGCGGTGAT ACTGAGCACA T ¹⁴¹ (1) data for SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 147 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

CGATGTGCTC

ACCGCAGATG

TTGGTAGGTG

AGTATCACCG

GTTATCTGTA

AGAGCGGAAT

CCAGTGGTAT

TGTTTTTTAT

TAGACGT

TTATGTCAAC ACCGCCAGAG ATAATTTATC

ATGAATTTAT TTTTTGCAGG GGGGCATTGT

120

147

data to SEQ NO ID: 55: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 55 basic pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: j simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC GGTAC 55 (1) data for SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs

01-1718-97 Ce • · · ·

173 (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

CGAATTCCAA CGCGTTAACC ATATGTTATT CCTCCTTCTA GAATCAAAT 49 (1) data for SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 668 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

GTGAAGAGCG TGAAGAGCGG TTCCTCCTTT CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA 60 GGCCCCAAGG GGTTATGCTA GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT 120 TCGGGCTTTC TTČTTCTTCT TCTTCTTTCC GCGGATCCTC GAGTAAGCTT CCATGGTACC 180 CTGCAGGTCG ACACTAGTGA GCTCGAATTC CAACGCGTTA ACCATATGTT ATTCCTCCTT 240 TAATTAGTTA ACTCAAATCT AGAATCAAAT CGATAAATTG TGAGCGCTCA CAATTGAGAA 300 TATTAATCAA GAATTTTAGC ATTTGTCAAA TGAATTTTTT AAAAATTATG AGACGTCCAT 360 ATTTGAATGT ATTTAGAAAA ATAAACAAAA GAGTTTGTAG AAACGCAAAA AGGCCATCCG _p 420 TCAGGATGGC CTTCTGCTTA ATTTGATGCC TGGCAGTTTA TGGCGGGCGT CCTGCCCGCC 480 ACCCTCCGGG CCGTTGCTTC GCAACGTTCA aatccgctcc CGGCGGATTT GTCCTACTCA 540 GGAGAGCGTT CACCGACAAA CAACAGATAA AACGAAAGGC CCAGTCTTTC GACTGAGCCT 600 TTCGTTTTAT TTGATGCCTG GCAGTTCCCT ACTCTCGCAT GGGGAGACCA TGCATACGTT 660

ACGCACGT

668

01-1718-97 Ce

174 (1) data of SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 726 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

GCGTAACGTA TGCATGGTCT CCCCATGCGA GAGTAGGGAA CTGCCAGGCA TCAAATAAAA 60 CGAAAGGCTC AGTCGAAAGA CTGGGCCTTT CGTTTTATCT GTTGTTTGTC GGTGAACGCT 120 CTCCTGAGTA GGACAAATCC GCCGGGAGCG GATTTGAACG TTGCGAAGCA ACGGCCCGGA 180 GGGTGGCGGG CAGGACGCCC GCCATAAACT GCCAGGCATC AAATTAAGCA GAAGGGGCCT 240 CCCACCGCCC GTCCTGCGGG CGGTATTTGA CGGTCCGTAG TTTAATTCGT CTTCGCCATC 300 CTGACGGATG GCCTTTTTGC GTTTCTACAA ACTCTTTTGT TTATTTTTCT AAATACATTC 360 AAATATGGAC GTCTCATAAT TTTTAAAAAA TTCATTTGAC AAATGCTAAA ATTCTTGATT 420 AATATTCTCA ATTGTGAGCG CTCACAATTT ATCGATTTGA TTCTAGATTT GTTTTAACTA 480 ATTAAAGGAG GAÁTAACATA TGGTTAACGC GTTGGAATTC GAGCTCACTA GTGTCGACCT 540 GCAGGGTACC ATGGAAGCTT ACTCGAGGAT CCGCGGAAAG AAGAAGAAGA AGAAGAAAGC 600 CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG 660 GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GGAACCGCTC TTCACGCTCT 720 TČAČGC 726 ! and (1) entry e to SEQ ID NO : 59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ·· · ♦ ·· ·· ·· · 9 · 9 9 9

9,999 · 9

99 9 999999

01-1718-97 Ce

175 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

TACGCACTGG ATCCTTATAA GCAGCTTATT TTTACTGATT 'GGAC' 44 (1) data for SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

GTCCTCCTGG TACCTACCTA AAACÁAC 27 (1) data for SEQ ID NO: 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTACCCGGCG 60

GACATTTATC ACACAGCAGC TGATGAGAAG TTTCTTCATC CA 102 (1) data for SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid

01-1718-97 Ce ·· ·· · · · · · · · ·

176 (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

Met Asp Glu Glu Thr Ser Gin Leu Leu Cys Asp Lys Cys Pro 1 5 10 15

Gly Thr Tyr (1) data for SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

TATGGAAACT TTCCTCCTCCAA AATATCTTCA TTATGATGAA GAAACTTCTC ATCAGCTGCT

GTGTGATAAA TGTCCGCCGG GTAC (1) data for SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs (Β) ΤΫΡ: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCATAA TGAAGATATT 60

TTGGAGGAAA AGTTTCCA ·· ··

01-1718-97 ftftft ftftftftftftftftftftftftftft ftftftft ftft © β β © 9

177 (1) data of SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

TACGCACTGG ATCCTTATAA GCAGCTTATT TTCACGGATT GAAC (1) data for SEQ ID NO: 66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

GTGCTCCTGG TACCTACCTA AAACAGCACT GCACAGTG (1) data for SEQ ID NO: 67:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ NO ID: 67:

01-1718-97 Če

178

TATGGAAACT CTGCCTCCAA AATACCTGCA TTACGATCCG GAAACTGGTC ATCAGCTGCT 60

GTGTGATAAA TGTGCTCCGG GTAC 84 (1) data for SEQ ID NO: 68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

CCGGAGCACA TTTATCACAC AGCAGCTGAT GYCCAGTTTC CGGATCGTAA TGCAGGTATT 60

TTGGAGGCAG AGTTTCCA 78 (1) data for SEQ ID NO: 69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

TATGGACCCA GAAACTGGTC ATCAGCTGCT GTGTGATAAA .TGTGCTCCGG_ ^ GTAC ___ 54 _ (1) data for SEQ ID NO: 70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA

01-1718-97 Jul _ ·: * *. · · ·· :::

·! ·· ··· ··· ···· ·· ··

179 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:

CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC TGGGTCCA 48 (1) data for SEQ ID NO: 71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

TATGAAAGA ACTCTGCCTC CAAAATACCT GCATTACGAT CCGGAAACTG GTCATCAGCT 60

GCTGTGTGAT AAATGTGCTC CGGGTAC 87 (1) data for SEQ ID NO: 72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA TGCAGGTATT 60

TTGGAGGCAG AGTTTCTTTC A 81 (1) data for SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

01-1718-97 About · · ·· · · · ·

180 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:

GTTCTCCTCA TATGAAACAT CATCACCATC ACCATCAIGA AACTCTGCCT CCAAAAIACC TGCATTACGA T (1) data for SEQ ID NO: 7 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:

GTTCTCCTCA TATGAAAGAA ACTCTGCCTC CAAAATACCT GCA (1) data for SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:

TACGCACTGG ATCCTTAATG ATGGTGATGGTGATGATGTA AGCAGCTTAT TTTCACGGAI

TGAACCTGAT TCCCTA (1) data for SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid

01-1718-97 · · · ♦ · ·· .1 ί

181 (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

GTTCTCCTCA TATGAAATAC CTGCATTACG ATCCGGAAAC TGGTCAT (1) data for SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:

GTTCTCCTAT TAATGAAATA TCTTCATTAT GATGAAGAAA CTT 43 (1) data for SEQ ID NO: 78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear _ (ii) MOLECULAR TYPE: cDNA ^r (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:

TACGCACTGG

ATCCTTATAA GCAGCTTATT TTTACTGATT (1) data for SEQ ID NO: 79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

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182 (B) TYPE: nucleic acid (C) CHAIN TYPE: single

TOPOLOGY: linear _ (ii) MOLECULAR TYPE: cDNA · · ..........

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:

GTTCTCCTCA TATGGAAACT CTGCCTCCAA AATACCTGCA (1) data for SEQ ID NO: 80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:

TACGCACTGG ATCCTTATGT TGCATTTCCT TTCTGAATTA GCA (1) data for SEQ ID NO: 81:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:

CCGGAAACAG ATAATGAG (1) data for SEQ ID NO: 82:

(i) SEQUENCE CHARACTERISTICS:

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183 (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:

GATCCTCATT ATCTGTTT 18 (1) data for SEQ ID NO: 83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:

CCGGAAACAG AGAAGCCACG CAAAAGTAAG 30 (1) data for SEQ ID NO: 84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:

GATCCTTACT TTTGCGTGGC TTCTCTGTTT 30 (1) data for SEQ ID NO: 85:

• · 9

01-1718-97 Ce

184 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear - J i

(ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:

TATGTTAATG AG 12 (1) data for SEQ ID NO: 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:

GATCCTCATT AACA 14 (1) data for SEQ ID NO: 87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid

.......... (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:

TATGTTCCGG AAACAGTTAA G 21 (1) data for SEQ ID NO: 88:

01-1718-97 Ce

185 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear i

(ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:

GATCCTTAAC TGTTTCCGGA ACA 23 (1) data for SEQ ID NO: 89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:

TATGTTCCGG AAACAGTGAA TCAACTCAAA AATAAG 3 6 (1) data of SEQ ID NO: 90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:

GATCCTTATT TTTGAGTTGA TTCACTGTTT CCGGAACA 38 (1) data for SEQ ID NO: 91:

01-1718-97 Ce

186 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 100 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:

CTAGCGACGA CGACGACAAA GAAACTCTGC CTCCAAAATA CCTGCATTAC

CTGGTCATCA GCTGCTGTGT GATAAATGTG CTCCGGGTAC

GATCCGGAAA

100 (1) data of SEQ ID NO: 92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:

CCGGAGCACA TTTATCACAC AGCAGCTGAT GACCAGTTTC CGGATCGTAA

TTGGAGGCAG AGTTTCTTTG TCGTCGTCGT CG

TGCAGGTATT (1) data for SEQ ID NO: 93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:

ACAAACACAA TGCATTTGAT ACTAGA

01-1718-97 Ce * ·

187 (1) data of SEQ ID NO: 94:

(i) SEQUENCE CHARACTERISTICS:

- - (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:

TTTGTTTTAA CTAATTAAAG GAGGAATAAA ATATGAGAGG ATCGCATCAC 50 (1) data for SEQ ID NO: 95:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:

CATCACCATC ACGAAACCTT CCCGCCGAAA TACCTGCACT ACGACGAAGA 50 (1) data for SEQ ID NO: 96:

(i) SEQUENCE CHARACTERISTICS: “. (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:

01-1718-97 Ce

188

AACCTCCCAC CAGCTGCTGT GCGACAAATG CCCGCCGGGT ACCCAAACA 49 (1) Data on SEQ ID NO: 97:

(i) SEQUENCE CHARACTERISTICS: .........

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:

TGTTTGGGTA CCCGGCGGGC ATTTGT 26 (1) data for SEQ ID NO: 98:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:

CGCACAGCAG CTGGTGGGAG GTTTCTTCGT CGTAGTGCAG GTATTTCGGC 50 (1) data for SEQ ID NO: 99:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:

··

01-1718-97 Če

189

GGGAAGGTTT CGTGATGGTG ATGGTGATGC GATCCTCTCA TATTTTATT 49 (1) data for SEQ ID NO: 100:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:

CCTCCTTTAA TTAGTTAAAA CAAATCTAGT ATCAAATCGA TTGTGTTTGT 50 (1) data for SEQ ID NO: 101:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:

ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG 59 (1) Data on SEQ ID NO: 102:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA ··

01-1718-97 Ce

190 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:

CTAATTAAAG GAGGAATAAA ATGAAAGAAA CTTTTCCTCC AAAATATC 48 (1) data for SEQ ID NO: 103:

/ (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:

TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31 (1) data for SEQ ID NO: 104:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:

ACAAACACAA TCGATTTGAT ACTAGATTTG TTTTAACTAA TTAAAGGAGG AATAAAATG 59 (1) Data on SEQ ID NO: 105:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA

01-1718-97 Ce ··· ··················· · · · I ··· 9 ·

191 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:

CTAATTAAAG GAGGAATAAA ATGAAAAAAA AAGAAACTTT TCCTCCAAAA TATC 54 (1) data for SEQ ID NO: 106:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:

TGTTTGGGTA CCCGGCGGAC ATTTATCACA C 31

data for SEQ ID NO: 107: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 44 basic pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:

CAGCCCGGGT AAAATGGAAA CGTTTCCTCC AAAATATCTT CATT 44 (1) data for SEQ ID NO: 108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA

01-1718-97 · a a a a a -17 -17 -17 -17 -17 -17 -17 -17 -17 -17

192 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:

CGTTTCCATT TTACCCGGGC TGAGCGAGAG GCTCTTCTGC GTGT

data to SEQ NO ID: 109: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 45 basic pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:

CGCTCAGCCC GGGTAAAATG GAAACGTTGC CTCCAAAATA CCTGC (1) data for SEQ ID NO: 110:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:

CCATTTTAČC CGGGCTGAGC GAGAGGCTCT TCTGCGŤGT ⁼ ”(1) the data for SEQ ID NO: 111:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

01-1718-97 Če

193

ft (ft) ft ft ft ft (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111

GAAAATAAGC-TGCTTAGGG GAGCTGAACC AAAATC = 36 (1) data for SEQ ID NO: 112:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:

CAGCTGCAGC TAAGCAGCTT ATTTTCACGG ATTG 34 (1) data for SEQ ID NO: 113:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:

AAAAATAAGC TGCTTAGAG CAGCTGAACC AAAATC 36 (1) data for SEQ ID NO: 114:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear · 99 · 9 · 9 · 9 · ··· ·· ·· ··

01-1718-97 Ce β

··· 9

194 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:

CAGCTGCAGC TAAGCAGCTT ATTTTTACTG ATTGG - 35 (1) data of SEQ ID NO: 115:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:

CTAGAAGGAG GAATAACATA TGGAAACTTT TGCTCCAAAA TATCTTCATT ATGATGAAGA 60

AACTAGTCAT CAGCTGCTGT GTGATAAATG TCCGCCGGGT AC 102 (1) data for SEQ ID NO: 116:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA ”(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: - - ™ '

CCGGCGGACA TTTATCACAC AGCAGCTGAT GACTAGTTTC TTCATCATAA TGAAGATATT 60

TTGGAGCAAA AGTTTCCATA TGTTATTCCT CCTT (1) data for SEQ ID NO: 117:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid ··

01-1718-97 Ce «··· ♦ ·· ·· -. 9 · 9 9 9 9

9 99 ·· · e · · · · · ·

195 (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: - /

CTAGAAGGAG GAATAACATA TGGAAACTTT TCCTGCTAAA TATCTTCATT ATGATGAAGA 60

AA 62 (1) data of SEQ ID NO: 118:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:

CTAGTTTCTT CATCATAATG AAGATATTTA GCAGGAAAAG TTTCCATATG TTATTCCTCC 60

TT 62 (1) data for SEQ ID NO: 119:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:

· »• ·

01-1718-97 English • · ··

Tyr 1 His Tyr Tyr Asp 5 Gin Asn Gly Arg Cys Gin For Gly 20 May His Phe Leu Wall Lys 25 Asp Thr Wall Cys His Lys For Cys Glu

40

196

Met 10 Cys Glu Glu Cys His 15 Dec Met His Cys Lys Gin For 30 Lys Arg For Gly Wall Thr 45 Tyr Thr Asp t

Asp Trp His

data for SEQ ID NO: 120: (and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2432 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (ix) CHARACTER: (A) NAME / KEY: CDS (B) POSITION: 124..1326

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:

ATCAAAGGCA GGGCATACTT CCTGTTGCCC AGACCTTATA

GGCAGCAGAG AAGCACCTAG CACTGGCCCA GCGGCTGCCG

ACA ATG AAC AAG TGG CTG TGC TGT

Met Asn Lys Trp Leu

10

ATT GAA GGG ACA ACC CAG

Ile Glu Thr Thr Gin Glu Thr Phe Pro

25

TAAAACGTCA TGTTCGCCTG

CCTGAGGTTT CCAGAGGACC

GTG TTC TTG GAC .ATC Wall Phe Leu Asp Ile 15 Dec AAA TRAY TTG CAT MELT Lys Tyr Leu His 30 Tyr

120

168.

216 ··

01-1718-9 / β β • • • • •

197

GAC CCA GAA Glu ACC GGA Thr Gly 35 CGT CAG CTC TTG TGT GAC AAA 264 Asp For Arg Gin Leu Leu Cys Asp Lys Cys Ala 45 For Gly ACC TRAY CTA AAA CAG CAC TGC ACA GTC AGG, AGG AAG ACA CTG TGT GTC, 312 Thr Tyr Leu Lys Gin His Cys Thr Wall Arg Arg Lys Thr Leu Cys Wall 50 55 60 l CCT TGC CCT GAC TRAY TCT MELT ACA GAC AGC TGG CAC ACG AGT GAT GAA 360 For Cys For Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu 65 70 75 TGC GTG TRAY TGC AGC CCC GTG TGC AAG GAA CTG CAG ACC GTG AAA CAG 408 Cys Wall Tyr Cys Ser For Wall Cys Lys Glu Leu Gin Thr Wall Lys Gin 80 85 90 95 GAG TGC AAC CGC ACC CAC AAC CGA GTG TGC GAA TGT GAG GAA GGG CGC 456 Glu Cys Asn Arg Thr His Asn Arg Wall Cys Glu Cys Glu Glu Gly Arg 100 ALIGN! 105 110 TRAY CTG GAG CTC GAA TTC TGC TTG AAG CAC CGG AGC TGT CCC CCA GGC 504 Tyr Leu Glu Leu Glu Phe Cys Leu Ly3 His Arg Ser Cys For For Gly 115 120 125 TTG GGT GTG CTG CAG GCT GGG ACC CCA GAG CGA AAC ACG GTT TGC AAA 552 Leu Gly Wall Leu Gin Ala Gly Thr For Glu Arg Asn Thr Wall Cys Lys 130 135 140 AGA TGT CCG GAT GGG TTC TTC TCA GGT GAG ACG TCA TCG AAA GCA CCC 600 Arg Cy3 For Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala For 145 150 155 TGT AGG AAA CAC ACC AAC TGC AGC TCA CTT GGC CTC CTG CTA ATT CAG 648 Cys Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu Ile Gin 160 165 170 175 AAA GGA AAT GCA ACA CAT GAC AAT GTA TGT TCC GGA AAC AGA GAA GCA 696 Lys Gly Asn Ala Thr His Asp Asn Wall Cys Ser Gly Asn Arg Glu Ala 180 185 190 ACT CAA ’ AAT TGT GGA ΆΤΑ ” GAT GTC ACC CTG TGC ’ GAA GAG GCA TTC " ttc - 744 Thr Gin Asn Cys Gly ile Asp Wall Thr Leu Cys Glu Glu Ala Phe Phe 195 200 205 AGG TTT GCT GTG CCT ACC AAG ATT ATA CCG AAT TGG CTG AGT GTT CTG 792 Arg Phe Ala Wall For Thr Lys Ile Ile For Asn Trp Leu Ser Wall Leu 210 215 220 GTG GAC AGT TTG CCT GGG ACC AAA GTG AAT GCA GAG AGT GTA GAG AGG 840 Wall Asp Ser Leu For Gly Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg 225 230 235

• ·

01-1718-97 '

198

ATA AAA AGG CAC AGC TCG CAA Gin GAG CAA ACT TTC CAG 888 Ile Lys 240 Arg Arg His Ser 245 Ser Glu Gin Thr 250 Phe Gin Leu Leu Lys 255 CTG TGG AAG CAT CAA AAC AGA GAC CAG GAA ATG GTG AAG AAG ATC ATC 936 Leu Trp Lys His Gin Asn Arg Asp Gin Glu Met Wall Lys Lys Ile Ile 260 265 270 CAA GAC ATT GAC CTC TGT GAA AGC AGT GTG CAA CGG CAT ATC GGC CAC 984 Gin Asp Ile Asp Leu Cys Glu Ser Ser Wall Gin Arg His Ile Gly His 275 280 285 GCG AAC CTC ACC ACA GAG CAG CTC CGC ATC TTG ATG GAG AGC TTG CCT 1032 Ala Asn Leu Thr Thr Glu Gin Leu Arg Ile Leu Met Glu Ser Leu For 290 295 300 GGG AAG AAG ATC AGC CCA GAC GAG ATT GAG AGA ACG AGA AAG ACC TGC 1080 Gly Lys Lys Ile Ser For Asp Glu ile Glu Arg Thr Arg Lys Thr Cys 305 310 315 AAA CCC AGC GAG CAG CTC CTG AAG CTA CTG AGC TTG TGG AGG ATC AAA 1128 Lys For Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys 320 325 330 335 AAT GGA GAC CAA GAC ACC TTG AAG GGC CTG ATG TRAY GCA CTC AAG CAC 1176 Asn Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His 340 345 350 TTG AAA GCA TRAY CAC TTT CCC AAA ACC GTC ACC CAC AGT CTG AGG AAG 1224 Leu Lys Ala Tyr His Phe For Lys Thr Wall Thr His Ser Leu Arg Lys 355 360 365 ACC ATC AGG TTC TTG CAC AGC TTC ACC ATG TRAY CGA TTG MELT CAG AAA 1272 Thr Ile Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys 370 - 375 380 CTC TTT CTA GAA ATG ATA GGG AAT CAG GTT CAA TCA GTG AAG ATA AGC 1320 Leu Phe Leu Glu Met Ile Gly Asn Gin Wall Gin Ser Wall Lys Ile Ser 385 390 395

TGC TTA TAGTTAGGAA TGGTCACTGG GCTGTTTCTT CAGGATGGGC CAACACTGAT 1376

Cys Leu 400 '!

GGAGCAGATG GCTGCTTCTC CGGCTCTTGA AATGGCAGTT GATTCCTTTC TCATCAGTTG 1436

GTGGGAATGA AGATCCTCCA GCCCAACACA CACACTGGGG AGTCTGAGTC AGGAGAGTGA 1496

GGCAGGCTAT TTGATAATTG TGCAAAGCTG CCAGGTGTAC ACCTAGAAAG TCAAGCACCC

1556 · β β β........

4

01-1718-97 Če

β ··

199

TGAGAAAGAG GATATTTTTA TAACCTCAAA CATAGGCCCT TTCCTTCCTC TCCTTATGGA 1616 TGAGTACTCA GAAGGCTTCT ACTATCTTCT GTGTCATCCC TAGATGAAGG CCTCTTTTAT 1676 TTATTTTTTT ATTCTTTTTT TCGGAGCTGG GGACCGAACC CAGGGCCTTG CGCTTGCGAG ’1736 GCAAGTGCTC TACCACTGAG CTAAATCTCC AACCCCTGAA GGCCTCTTTC TTTCTGCCTC 1796 TGATAGTCTA TGACATTCTT TTTTCTACAA TTCGTATCAG GTGCACGAGC CTTATCCCAT 1856 TTGTAGGTTT CTAGGCAAGT TGACCGTTAG CTATTTTTCC CTCTGAAGAT TTGATTCGAG 1916 TTGCAGACTT GGCTAGACAA GCAGGGGTAG GTTATGGTAG TTTATTTAAC AGACTGCCAC 1976 CAGGAGTCCA GTGTTTCTTG TTCCTCTGTA GTTGTACCTA AGCTGACTCC AAGTACATTT 2036 AGTATGAAAA ATAATCAACA AATTTTATTC CTTCTATCAA CATTGGCTAG CTTTGTTTCA 2096 GGGCACTAAA AGAAACTACT ATATGGAGAA AGAATTGATA TTGCCCCCAA CGTTCAACAA 2156 CCCAATAGTT TATCCAGCTG TCATGCCTGG TTCAGTGTCT ACTGACTATG CGCCCTCTTA 2216 TTACTGCATG CAGTAATTCA ACTGGAAATA GTAATAATAA TAATAGAAÁT AAAATCTAGA 2276 CTCCATTGGA TCTCTCTGAA TATGGGAATA TCTAACTTAA GAAGCTTTGA GATTTCAGTT 2336 GTGTTAAAGG CTTTTATTAA AAAGCTGATG CTCTTCTGTA AAAGTTACTA ATATATCTGT 2396 AAGACTATTA CAGTATTGCT ATTTATATCC ATCCAG 2432

(1) data for SEQ ID NO-121:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 401 amino acids, ... (B) TYPE: amino acid - "(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein /

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:

Met Asn Lys Trp Leu Cys Cys Ala Leu Leu Le Phu Leu Asp Ile Ile ¹ 5 10 15

Glu Trp Thr Thr Gin Glu Thr Phe Pro Lys Tyr Leu His Tyr Asp 20 25 30 ·· ·· • ·

01-1718-97 Če -17 -17 -17 -17 -17 -17 -17 -17 -17 e e * * * * *

9 ·· · ··

200

Pro Glu Thr Gly Arg For Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Wall Arg Arg Lys Thr Leu Cys Wall For 50 55 60 Cys For Asp Tyr Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Wall Tyr Cys Ser For Wall Cys Lys Glu Leu Gin Thr Wall Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Wall Cys Glu Cys Glu Glu Gly Arg Tyr 100 ALIGN! 105 110 Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cys For For Gly Leu 115 120 125 Gly Wall Leu Gin Ala Gly Thr For Glu Arg Asn Thr Wall Cys Lys Arg 130 135 140 Cys For Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala For Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Ser Leu Gly Leu Leu Leu Ile Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Wall Cys Ser Gly Asn Arg Glu Ala Thr 180 185 190 Gin Asn Cys Gly Ile Asp Wall Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Wall For Thr Lys Ile Ile For Asn Trp Leu Ser Wall Leu Wall 210 - 215 220 Asp Ser Leu For Gly Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg Ile 225 230 235 240 Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp you with His Gin Asn Arg Asp Gin Glu Met Wall Lys Lys Ile Ile Gin 260 265 270 Asp Ile Asp Leu Cys Glu Ser Ser Wall Gin Arg His Ile Gly His Ala

275 280 285

Asn Leu Thr Thr Glu Gin Leu Arg Ile Leu Met Glu Ser Leu Pro Gly 290 295 300 »»

01-1718-97 Ce

201 ·· · * β ββ «4 · · f · · Α · · ·

Lys 305 Lys Ile Ser For Asp 310 Glu Ile Glu Arg Thr 315 Arg Lys Thr Cys Lys 320 For Ser Glu Gin Leu 325 Leu Lys Leu Leu Ser 330 Leu Trp Arg Ile Lys 335 Asn Gly Asp Gin Asp 340 Thr Leu Lys Gly Leu 345 Met Tyr Ala Leu Lys 350 His Leu Lys Ala Tyr 355 His Phe For Lys Thr 360 Wall Thr His Ser Leu 365 Arg Lys Thr Ile Arg 370 Phe Leu His Ser Phe 375 Thr Met Tyr Arg Leu 380 Tyr Gin Lys Leu Phe 385 Leu Glu Met Ile Gly 390 Asn Gin Wall Gin Ser 395 Wall Lys Ile Ser Cys 400

Leu (1) data for SEQ ID NO: 122:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1324 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (ix) SIGN:

(A) NAME / KEY: CDS (B) POSITION: 90..1292 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:

CCTTATATAA ACGTCATGAT TGCCTGGGCT GCAGAGACGC ACCTAGCACT GACCCAGCGG 60

CTGCCTCCTG AGGTTTCCCG AGGACCACA ATG AAC AAG TGG

Met Asn Lys Trp

5

CTC CTG GTG CTC CTG GAC ATC ATT GAA TGG ACA ACC CAG GAA ACC CTT Leu Leu Leu Leu Asp Ile Ile Glu Thr Thr Gin Glu Thr Leu 10 15 20

161

I e e

01-1718-97 Ce

202

CCT CCA Pro Pro 25 AAG Ly3 TRAY Tyr TTG Leu CAT His TAT GAC CCA GAA ACT GGT CAT His CAG Gin CTC Leu CTG Leu 40 209 Tyr Asp For Glu Thr 35 Gly TGT GAC AAA TGT GCT CCT GGC ACC TRAY CTA AAA CAG CAC TGC ACA GTG '257 Cys Asp Lys Cys Ala For Gly Thr Tyr Leu Lys Gin His Cys Thr Wall 45 50 55 AGG AGG AAG ACA TTG TGT GTC CCT TGC CCT GAC CAC TCT MELT ACG GAC 305 Arg Arg Lys Thr Leu Cys Wall For Cys For Asp His Ser Tyr Thr Asp 60 65 70 AGC TGG CAC ACC AGT GAT GAG TGT GTG MELT TGC AGC CCA GTG TGC AAG 353 Ser Trp His Thr Ser Asp Glu Cys Wall Tyr Cys Ser For Wall Cys Lys 75 80 85 GAA CTG CAG TCC GTG AAG CAG GAG TGC AAC CGC ACC CAC AAC CGA GTG 401 Glu Leu Gin Ser Wall Lys Gin Glu Cys Asn Arg Thr His Asn Arg Wall 90 95 100 ALIGN! TGT GAG TGT GAG GAA GGG CGT TRAY CTG GAG ATC GAA TTC TGC TTG AAG 449 Cys Glu Cys Glu Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu Lys 105 110 115 120 CAC CGG AGC TGT CCC CCG GGC TCC GGC GTG GTG CAA GCT GGA ACC CCA 497 His Arg Ser Cys For For Gly Ser Gly Wall Wall Gin Ala Gly Thr For 125 130 135 GAG CGA AAC ACA GTT TGC AAA AAA TGT CCA GAT GGG TTC TTC TCA GGT 545 Glu Arg Asn Thr Wall Cys Lys Lys Cys For Asp Gly Phe Phe Ser Gly 140 145 150 GAG. ACT TCA TCG AAA GCA CCC TGT ATA AAA CAC ACG AAC TGC AGC ACA 593 Glu Thr Ser Ser Lys Ala For Cys Ile Lys His Thr Asn Cys Ser Thr 155 160 165 TTT GGC CTC CTG CTA ATT CAG AAA GGA AAT GCA ACA CAT GAC AAC GTG 641 Phe Gly Leu Leu Leu Ile Gin Lys Gly Asn Ala Thr His Asp Asn Wall = -r. ~ ·: · -I -170- 175 18 Ó TGT TCC GGA AAC AGA GAA GCC ACG CAA AAG TGT GGA ATA GAT GTC ACC 689 Cys Ser Gly Asn Arg Glu Ala Thr Gin Lys Cys Gly Ile Asp Wall Thr 185 190 195 200 CTG TGT GAA GAG GCC TTC TTC AGG TTT GCT GTT CCT ACC AAG ATT ATA 737 Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Wall For Thr Lys Ile Ile 205 210 215

203

CCA AAT TGG GTG GTG TTG GTG GTG GCT 785 For Asn Trp Leu 220 Ser Val Leu Val Asp 225 Ser Leu Pro Gly Thr 230 Lys Val AAT GCC GAG AGT GTA GAG AGG ATA AAA CGG AGA CAC AGC TCA CAA GAG 833 Asn Ala Glu Ser Wall Glu Arg Ile Lys Arg Arg His Ser Ser Gin Glu 235 240 245 CAA ACC TTC CAG CTG CTG AAG CTG TGG AAA CAT CAA AAC AGA GAC CAG 881 Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Arg Asp Gin 250 255 260 GAA ATG GTG AAG AAG ATC ATC CAA GAC ATT GAC CTC TGT GAA AGC AGC 929 Glu Met Wall Lys Lys Ile Ile Gin Asp Ile Asp Leu Cys Glu Ser Ser 265 270 275 280 GTG CAG CGG CAT CTC GGC CAC TCG AAC CTC ACC ACA GAG CAG CTT CTT 977 Wall Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu Gin Leu Leu 285 290 295 GCC TTG ATG GAG AGC CTG CCT GGG AAG AAG ATC AGC CCA GAA GAG ATT 1025 Ala Leu Met Glu Ser Leu For Gly Lys Lys Ile Ser For Glu Glu Ile 300 305 310 GAG AGA ACG AGA AAG ACC TGC AAA TCG AGC GAG CAG CTC CTG AAG CTA 1073 Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu Leu Lys Leu 315 320 325 CTC AGT TTA TGG AGG ATC AAA AAT GGT GAC CAA GAC ACC TTG AAG GGC 1121 Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gin Asp Thr Leu Lys Gly 330 335 340 CTG ATG MELT GCC CTC AAG CAC TTG AAA ACA TCC CAC TTT CCC AAA ACT 1169 Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe For Lys Thr 345 350 355 360 GTC ACC CAC AGT CTG AGG AAG ACC ATG AGG TTC CTG CAC AGC TTC ACA 1217 Wall Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His Ser Phe Thr - .365 370 - · ·· =. · 375 ATG TRAY AGA CTG MELT CAG AAG CTC TTT TTA GAA ATG ATA GGG AAT CAG 1265 Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met Ile Gly Asn Gin ! 380 385 390 GTT CAA TCC GTG AAA ATA AGC TGC TTA TAACTAGGAA TGGTCACTGG 1312 Wall Gin Ser Wall Lys Ile Ser Cys Leu 395 400

GCTGTTTCTT CA

1324 ··>

204 (1) data of SEQ ID NO: 123:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 401 amino acids (B) (D) TYPE: amino acid TOPOLOGY: linear (ii) SPECIES MOLECULES: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123

Met 1 Asn Lys Trp Leu Cys Cys Ala Leu Leu Val Leu Leu Asp Ile 15 Dec Ile 5 10 Glu Trp Thr Thr Gin Glu Thr Leu For For Lys Tyr Leu His Tyr Asp 20 May 25 30 For Glu Thr Gly His Gin Leu Leu Cys Asp Lys Cys Ala For Gly Thr 35 40 45 Tyr Leu Lys Gin His Cys Thr Wall Arg Arg Lys Thr Leu Cys Wall For 50 55 60 Cys For Asp His Ser Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys 65 70 75 80 Wall Tyr Cys Ser For Wall Cys Lys Glu Leu Gin Ser Wall Lys Gin Glu 85 90 95 Cys Asn Arg Thr His Asn Arg Wall Cys Glu Cys Glu Glu Gly Arg Tyr 100 ALIGN! 105 110 Leu Glu Ile Glu Phe Cys Leu Lye His Arg Ser Cys For For Gly Ser 115 - 120 125 Gly Wall Wall Gin Ala Gly Thr For Glu Arg Asn Thr Wall Cys Lys Lys 130 135 140 ....... Cys For Asp Gly Phe Phe Ser Gly Glu Thr Ser Ser Lys Ala For Cys 145 150 155 160 Ile / Lys His Thr Asn Cys Ser Thr Phe Gly Leu Leu Leu Ile Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Wall Cys Ser Gly Asn Arg Glu Ala Thr

180 185 190

Gin Lys Cly Gle Ile Asp Val Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205

205

Phe For Thr Lys Ile 215 Ile Pro Asn Trp Leu 220 Ser .Wall Leu Wall 210 Asp Ser Leu For Gly Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg Ile 225 230 235 240 Lys Arg Arg His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys His Gin Asn Arg Asp Gin Glu Met Wall Lys Lys Ile Ile Gin 260 265 270 Asp Ile Asp Leu Cys Glu Ser Ser Wall Gin Arg His Leu Gly His Ser 275 280 285 Asn Leu Thr Thr Glu Gin Leu Leu Ala Leu Met Glu Ser Leu For Gly 290 295 300 Lys Lye Ile Ser For Glu Glu Ile Glu Arg Thr Arg Lys Thr Cys Lys 305 310 315 320 Ser Ser Glu Gin Leu Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu 340 345 350 Lys Thr Ser His Phe For Lys Thr Wall Thr His Ser Leu Arg Lys Thr 355 360 365 Met Arg Phe Leu His Ser Phe Thr Met Tyr Arg Leu Tyr Gin Ly3 Leu 370 375 380 Phe Leu Glu Met Ile Gly Asn Gin Wall Gin Ser Wall Lys Ile Ser Cys 385 - 390 395 400

Leu (1) data for SEQ ID NO: 124:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1355 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single ft ··· »

206 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (ix) SIGN:

( _A) NAME / KEY: CDS '' (B) POSITION: 94..1296 '(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:

GTATATATAA CGTGATGAGC GTACGGGTGC GGAGACGCAC CGGAGCGCTC GCCCAGCCGC 60

CGCTCCAAGC CCCTGAGGTT TCCGGGGACC ACA ATG AAC TTG CTG TGC TGC 114

Met Asn Lys Leu Leys Cys Cys

GCG CTC GTG TTT CTG GAC ATC 1 TCC ATT AAG TGG ACC 5 ACC CAG GAA ACG 162 Ala Leu Wall 10 Phe Leu Asp Ile Ser 15 Dec Ile Lys Trp Thr Thr 20 May Gin Glu Thr TTT CCT CCA AAG TRAY CTT CAT MELT GAC GAA GAA ACC TCT CAT CAG CTG 210 Phe For For Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His Gin Leu 25 30 35 TTG TGT GAC AAA TGT CCT CCT GGT ACC TRAY CTA AAA CAA CAC TGT ACA 258 Leu Cys Asp Lys Cys For For Gly Thr Tyr Leu Lys Gin His Cys Thr 40 45 50 55 GCA AAG TGG AAG ACC GTG TGC GCC CCT TGC CCT GAC CAC TRAY TRAY ACA 306 Ala Lys Trp Lys Thr Wall Cys Ala For Cys For Asp His Tyr Tyr Thr 60 65 70 GAC AGC TGG CAC ACC AGT GAC GAG TGT CTA TRAY TGC AGC CCC GTG TGC 354 Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser For Wall Cys 75 80 85 AAG GAG CTG CAG TRAY GTC AAG CAG GAG TGC AAT CGC ACC CAC AAC CGC 402 Lys Glu Leu Gin Tyr Wall Lys Gin Glu Cys Asn Arg Thr His Asn Arg 90 95 100 ALIGN! I = L --------- = === ^ - = · « = • i— · ^: = "- - ----- GTG TGC GAA TGC AAG GAA GGG CGC TRAY CTT GAG ATA GAG TTC TGC TTG 450 Wall Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe Cys Leu 105 110 115 AAA ČAT AGG AGC TGC CCT CCT GGA TTT GGA GTG GTG CAA GCT GGA ACC 498 Lys His Arg Ser Cys For For Gly Phe Gly Wall Wall Gin Ala Gly Thr 120 125 130 135 CCA GAG CGA AAT ACA GTT TGC AAA AGA TGT CCA GAT GGG TTC TTC TCA 546 For Glu Arg Asn Thr Wall Cys Lys Arg Cys For Asp Gly Phe Phe Ser 140 145 150

e e ···

207

01-1718-9 / English ► ···

AAT Asn GAG ACG TCA TCT AAA Lys GCA CCC TGT ACA AAT TGC AGT 594 Glu Thr Ser 155 Ser Ala For Cys Arg Lys 160 His Thr Asn 165 Cys Ser GTC TTT GGT CTC CTG CTA ACT CAG AAA GGA AAT GCA ACA CAC GAC AAC 642 Wall Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His Asp Asn 170 175 180

ATA TGT TCC GGA ATA GAT GTT 690 Ile Cys Ser Gly Asn Ser Glu 190 Ser Thr Gin Lys Cys 195 Gly Ile Asp Wall 185 ACC CTG TGT GAG GAG GCA TTC TTC AGG TTT GCT GTT CCT ACA AAG TTT 738 Thr Leu Cy3 Glu Glu Ala Phe Phe Arg Phe Ala Wall For Thr Lys Phe 200 205 210 215 ACG CCT AAC TGG CTT AGT GTC TTG GTA GAC AAT TTG CCT GGC ACC AAA 786 Thr For Asn Trp Leu Ser Wall Leu Wall Asp Asn Leu For Gly Thr Lys 220 225 230 GTA AAC GCA GAG AGT GTA GAG AGG ATA AAA CGG CAA CAC AGC TCA CAA 834 Wall Asn Ala Glu Ser Wall Glu Arg Ile Lys Arg Gin His Ser Ser Gin 235 240 245 GAA CAG ACT TTC CAG CTG CTG AAG TTA TGG AAA CAT CAA AAC AAA GCC 882 Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn Lys Ala 250 255 260 CAA GAT ATA GTC AAG AAG ATC ATC CAA GAT ATT GAC CTC TGT GAA AAC 930 Gin Asp Ile Wall Lys Lys Ile Ile Gin Asp Ile Asp Leu Cys Glu Asn 265 270 275 AGC GTG CAG CGG CAC ATT GGA CAT GCT AAC CTC ACC TTC GAG CAG CTT 978 Ser Wall Gin Arg His Ile Gly His Ala Asn Leu Thr Phe Glu Gin Leu 280 285 290 295 CGT agc TTG _ ATG GAA AGC TTA CCG GGA AAG 'AAA GTG GGA GCA GAA GAC 1026 Arg Ser Leu Met Glu Ser Leu For Gly Lys Lys Wall Gly Ala Glu Asp 300 305 310 ATT GAA AAA ACA ATA AAG GCA TGC AAA CCC AGT GAC CAG ATC CTG AAG 1074 Ile Glu Lys Thr ile Lys Ala Cys Lys For Ser Asp Gin Ile Leu Lys 315 320 325 CTG CTC AGT TTG TGG CGA ATA AAA AAT GGC GAC CAA GAC ACC TTG AAG 1122 Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gin Asp Thr Leu Lys

330 335 340 e · e β e 9 9 «s 9

01-1718-97 Eng · · · · · · · · · · · · · · · · · · · · · ·

208

GGC CTA by Gly Leu ATG CAC GCA Ala CTA Leu AAG CAC TCA Ser AAG ACG TAC CAC TTT CCC AAA 1170 Met His Lys 350 His Ly3 Thr Tyr 355 His Phe For Lys 345 ACT GTC ACT CAG AGT CTA AAG AAG ACC ATC AGG TTC CTT CAC AGC TTC 1218 Thr Wall Thr Gin Ser Leu Lys Lys Thr Ile Arg Phe Leu His Ser Phe and 360 365 370 375 '· ACA ATG TRAY AAA TTG MELT CAG AAG TTA TTT TTA GAA ATG ATA GGT AAC 1266 Thr Met Tyr Ly3 Leu Tyr Gin Lys Leu Phe Leu Glu Met Ile Gly Asn 380 385 390 CAG GTC CAA TCA GTA AAA ATA AGC TGC TTA TAACTGGAAA TGGCCATTGA 1316 Gin Wall Gin Ser Wall Lys Ile Ser Cys Leu

395 400

GCTGTTTCCT CACAATTGGC GAGATCCCAT GGATGATAA 1355 (1) data for SEQ ID NO: 125:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 401 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:

Met 1 Asn Lys Leu Leu 5 Cys Cys Ala Lys Trp Thr Thr 20 May Gin Glu Thr Phe Glu Glu Thr 35 Ser His Gin Leu Leu 40 Tyr Leu 50 Lys Gin His Cys Thr 55 Ala Cys 65 For Asp His Tyr Tyr 70 Thr Asp Leu Tyr Cys Ser For 85 Wall Cys Lys Cys Asn Arg Thr His Asn Arg Wall

100 ALIGN!

Leu Wall 10 Phe Leu Asp Ile Ser 15 Dec Ile For For Lys Tyr Leu His Tyr Asp 25 30 Cys Asp Lys Cys For 45 For Gly Thr Lys Trp Lys Thr 60 Wall Cys Ala For Ser Trp His 75 Thr Ser Asp Glu Cys 80 Glu Leu 90 Gin Tyr Wall Lys Gin 95 Glu Cys Glu Cys Lys Glu Gly Arg Tyr

105 110

01-1718-97 Če

209

Leu Glu Phe Cys Leu Lys His Arg. Ser Cys For 125 For Gly Phe 115 Gly Wall Wall Gin Ala Gly Thr For Glu Arg Asn Thr Wall Cys Lys Arg 130 135 140 Cys For Asp Gly Phe Phe Ser Asn Glu Thr Ser Ser Lys Ala For Cys 145 150 155 160 Arg Lys His Thr Asn Cys Ser Wall Phe Gly Leu Leu Leu Thr Gin Lys 165 170 175 Gly Asn Ala Thr His Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr 180 185 190 Gin Lys Cys Gly Ile Asp Wall Thr Leu Cys Glu Glu Ala Phe Phe Arg 195 200 205 Phe Ala Wall For Thr Lys Phe Thr For Asn Trp Leu Ser Wall Leu Wall 210 215 220 Asp Asn Leu For Gly Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg Ile 225 230 235 240 Lys Arg Gin His Ser Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu 245 250 255 Trp Lys His Gin Asn Lys Ala Gin Asp Ile Wall Lys Lys Ile Ile Gin 260 265 270 Asp Ile Asp Leu Cys Glu Aen Ser Wall Gin Arg His Ile Gly His Ala 275 280 285 Asn Leu Thr Phe Glu Gin Leu Arg Ser Leu Met Glu Ser Leu For Gly 290 295 300 Lys Lys Wall Gly Ala Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys 305 310 315 320 For Ser Asp Gin. Ile_ _r Leu. Lys_ Leu Leu. Ser Leu -Trp Arg Ile- Lys ’ 'Asn 325 330 335 Gly Asp Gin Asp Thr Leu Lys Gly Leu Met His Ala Leu Lys His Ser and 340 345 350 Lys Thr Tyr His Phe For Lys Thr Wall Thr Gin Ser Leu Lys Lys Thr 355 360 365 Ile Arg Phe Leu His Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu

370 375 380

01-1718-97 Che · «• φ

210

Phe Leu Glu Met Ile Gly Asn Gin Val Gin Ser Val Lys Ile Ser Cys ³⁸⁵ 390 395 ₄₀₀ Leu (1) Data on SEQ ID NO: 126:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 139 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:

Cys 1 For Gin Gly Lys 5 Tyr Ile His For Gin 10 Asn Asn Ser Ile Cys 15 Dec Cys Thr Lys Cys His 20 May Lys Gly Thr Tyr Leu 25 Tyr Asn Asp Cys For 30 Gly For Gly Gin Asp 35 Thr Asp Cys Arg Glu 40 Cys Glu Ser Gly Ser 45 Phe Thr Ala Ser Glu 50 Asn His Leu Arg His 55 Cys Leu Ser Cys Ser 60 Lys Cys Arg Lys Glu 65 Met Gly Gin Wall Glu 70 Ile Ser Ser Cys Thr 75 Wall Asp Arg Asp Thr 80 Wall -Cys- Gly Cys Arg- Lys85 Asn Gin 'TyrArg 90 His Tyr ' Trp " Ser ' Glu 95 Asn Leu Phe Gin Cys 100 ALIGN! Phe Asn Cys Ser Leu 105 Cys Leu Asn Gly Thr 110 Wall His Leu Ser Cys Gin Glu Lys Gin Asn Thr Wall Cys Thr Cys His Ala Gly

115 120 125

Phe Phe Leu Arg

01-1718-97 Ce ♦ · ···· ··· · · · · · ···

211

data to SEQ NO ID: 127: (and) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 48 base pairs (B). TYPE: nucleic acid- (C) CHAIN TYPE: j simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:

CCGGCGGACA TTTATCACAC AGCAGCTGAT GAGAAGTTTC TTCATCCA (1) data for SEQ ID NO: 128:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 128:

Met 1 Leu Gly Ile Trp 5 Thr Leu Leu For Leu 10 Wall Leu Thr Ser Wall 15 Dec Ala Arg Leu Ser Ser Lys Ser Wall Asn Ala Gin Wall Thr Asp Ile Asn Ser 20 May 25 30 Lys Gly / Leu 35 Glu Leu Arg Lys Thr 40 Wall Thr Thr Wall Glu 45 Thr Gin Asn Leu Glu 50 Gly Leu His His Asp 55 Gly Gin Phe Cys His 60 Lys For Cys For

Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 \ π π π X X

υ ± -ι / ± o-y / Ce «·

212 Asp Cys Wall For Cye Gin Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser Lys Cy3 Arg Arg Cys Arg Leu Cy3 Asp Glu Gly His Gly 100 ALIGN! 105 110 Leu Glu Wall Glu Ile Asn Cys Thr Arg Thr Gin Asn Thr Lys Cys Arg 115 120 125 Cys Lys For Aen Phe Phe Cys A3n Ser Thr Wall Cys Glu H3 Cys Asp 130 135 140 For Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150 155 160 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 165 170 175 Leu Cys Leu Leu Leu Leu For Ile For Leu Ile Wall Trp Wall Lys Arg 180 185 190 Lys Glu Wall Gin Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gin Gly 195 200 205 Ser His Glu Ser For Thr Leu Asn For Glu Thr

210 215 (1) data of SEQ ID NO: 129:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 280 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

(ii ) MOLECULAR TYPE: protein (xi ) “DESCRIPTION ^ SEQUENCE : SEQ ID NO: 129 Met Gly Leu Ser Thr Val Pro 4 Asp Leu Leu Leu For Leu Val Leu Leu 10 15 Glu Leu Leu Val Gly Ile Tyr Pro Gly Val Ile Gly Leu Val Pro 25 30 His Leu Gly 35 Asp Arg Glu Lys Arg Asp Ser Val Cys For Gin Gly Lys 40

01-1718-97 Če

213

Tyr Ile His Pro Gin Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50 55 Gly Thr Tyr Leu Tyr Asn Asp Cys For Gly For Gly Gin Asp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Glv Gin Wall 100 ALIGN! 105 110 Glu Ile Ser Ser Cys Thr Wall Asp Arg Asp Thr Wall Cys Gly Cys Arg 115 120 125 Lys Asn Gin Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gin Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn Gly Thr Wall His Leu Ser Cys Gin Glu 145 150 155 160 Lys Gin Asn Thr Wall Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 Asn Glu Cys Wall Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180 185 190 Lys Leu Cys Leu For Gin Ile Glu Asn Wall Lys Gly Thr Glu Asp Ser 195 200 205 Gly Thr Thr Wall Leu Leu For Leu Wall Ile Phe Phe Gly Leu Cys Leu 210 215 220 Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gin Arg Trp Lys 225 - 230 235 240 Ser Lys Leu Tyr Ser Ile Wall Cys Gly Lys Ser Thr For Glu Lys Glu : = _s - =,. .. .... 245 = .. .. 250 -, " 255 ,, - Gly Glu Leu Glu Gly Thr Thr Thr Lys For Leu Ala For Asn For Ser / 260 265 270 Phe Ser For Thr For Gly Phe Thr

275 280 · '· · ♦ ··· ♦ · <

• 9 9 9 9 9 9 9 99 99 99 99 99

01-1718-97 Ce

214 (1) data of SEQ ID NO: 130:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 207 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:

Met 1 Leu Arg Leu Ile 5 Ala Leu Leu Cys Val Val Tyr Gly 10 15 Dec Asp Asp Wall For Tyr Ser Ser Asn Gin Gly Lys Cys Gly Gly His Asp 20 May 25 30 Tyr Glu Lys Asp Gly Leu Cys Cys Ala Ser Cys His For Gly Phe Tyr 35 40 45 Ala Ser Arg Leu Cys Gly For Gly Ser Asn Thr Wall Cys Ser For Cys 50 55 60 Glu Asp Gly Thr Phe Thr Ala Ser Thr Asn His Ala For Ala Cys Wall 65 70 75 80 Ser Cys Arg Gly For Cys Thr Gly His Leu Ser Glu Ser Gin For Cy3 85 90 95 Asp Arg Thr His Asp Arg Wall Cys Asn Cys Ser Thr Gly Asn Tyr Cys 100 ALIGN! 105 110 Leu Leu Lys Gly Gin Asn Gly Cys Arg Ile Cys Ala For Gin Thr Lys 115 120 125 Cys For Ala Gly Tyr Gly Wall Ser Gly His Thr Arg Ala Gly Asp Thr 130 135 140 Leu Cys Glu Lys Cys For For His Thr Tyr Ser Asp Ser Leu Ser For 145 150 155 160 Thr Glu Arg Cys Gly Thr Ser Phe Asn Tyr Ile Ser Wall Gly Phe Asn

165 170 175

Leu Tyr Pro Val Asn Thr Ser Cys Thr Thr Thr Ala Gly His Asn 180 185 190

01 -1718-97 Če 215 · ' • ····  © · • '9 99 9 • « • Glu Wall Ile Lys 195 Thr Lys Glu Phe 200 Thr Wall Thr Leu Asn 205 Tyr Thr (1) Data on SEQ ID NO: 131: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 227 amino acids. (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: Met 1 Ala Pro Val Ala 5 Wall Trp Ala Ala Leu 10 Ala Wall Gly Leu Glu 15 Dec Leu Trp Ala Ala Ala His Ala Leu For Ala 25 Gin Wall Ala Phe Thr 30 For Tyr Ala For Glu Pro 35 Gly Ser Thr Cys 40 Arg Leu Arg Glu Tyr 45 Tyr Asp Gin Thr Ala 50 Gin Met Cys Cys Ser 55 Lys Cys Ser For Gly 60 Gin His Ala Lys Wall 65 Phe Cys Thr Lys Thr 70 Ser Asp Thr Wall Cys 75 Asp Ser Cys Glu Asp 80 Ser Thr Tyr Thr Gin 85 Leu Trp Asn Trp Wall 90 For Glu Cys Leu Ser 95 Cys Gly Ser Arg Cys Ser Ser Asp Gin Wall Glu Thr Gin Ala Cy3 Thr Arg .., 100 ALIGN! • s—- - = = · - - = · · -105 = ,, - - · - · - - - 110 · = -: = · - ·· - · Glu Gin Asn Arg Ile Cys Thr Cys 120 Arg For Gly Trp Tyr 125 Cys Ala Leu Ser Lys 130 Gin Glu Gly Cys Arg 135 Leu Cys Ala For Leu 140 Arg Lys Cys Arg For 145 Gly Phe Gly Wall Ala 150 Arg For Gly Thr Glu 155 Thr Ser Asp Wall Wall 160 Cys Lys Pro Cys Ala 165 For Gly Thr Phe Ser 170 Asn Thr Thr Ser Ser 175 Thr

01-1718-97 Če

216

4 © «

Asp Ile Cys Arg For His Gin Ile Cy3 Asn Wall Wall Ala Ile For Gly 180 185 190 Asn Ala Ser Arg Asp Ala Wall Cys Thr Ser Thr Ser For Thr Arg Ser 195 and.: ·. · _Ϊ.ϊ:. 200 205 Met Ala For Gly Ala Wall His Leu For Gin For Wall Ser Thr Arg Ser 210 215 220 Gin His Thr 225

(1) data e k SEQ NO ID: 132: (i) SEQUENCE CHARACTERISTICS: (AND) LENGTH: 197 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE MOLECULES: protein (xi)) SEQUENCE DESCRIPTION: SEQ ID NO: 132 Met Val Ser Leu For Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr 1 5 10 15 Dec Ala Val His Leu Gly Gin Cys Val Thr Cys Ser Asp Lye Gin Tyr Leu 20 May 25 30 His Asp Gly Gin Cys Cys Asp Leu Cys Gin For Gly Ser Arg Leu Thr 35 40 45 Ser .His Cys Thr , Ala ₌ Leu Glu, Ly3, Thr, Gin Cys .-His ’· For Cys Asp -Ser 50 55 60 Gly Glu Phe Ser Ala Gin Trp Asn Arg Glu Ile Arg Cys His Gin His 65 70 75 80 Arg His Cys Glu For Asn Gin Gly Leu Arg Wall Lys Lys Glu Gly Thr 85 90 95 Ala Glu Ser Asp Thr Val Cys Lys Glu Gly Gin His Cys Thr 100 ALIGN! 105 110 Ser Lys Asp Cys Glu Ala Cys Ala Gin His Thr For Cys Ile For Gly 115 120 125

9 · ·· · ♦ «9 9 9 9 9

01-1718-97 Če

217 • • β · 9

9 9

9 ··

Phe Gly Val Met Glu Met Ala Thr Glu Thr 136 Thr Asp Thr 140 Wall Cys His For Cys For Wall Gly Phe Phe Ser Asn Gin Ser Ser Leu Phe Glu Lys 145 150 155 160 Cys Tyr For Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Wall Leu Gin 165 170 175 Lys Gly Thr Ser Gin Thr Asn Wall Ile Cys Gly Leu Lys Ser Arg Met 180 185 190 Arg Ala Leu Leu Wall

195 (1) data of SEQ ID NO: 133:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 208 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:

Met Asn Lys Trp Leu Cys Ala Leu Leu Phu Leu Asp Ile Ile Asp 1 Glu Trp Thr 5 Thr Gin Glu Thr 10 Leu His 30 15 Dec Tyr Phe For 25 Pro Lys Tyr For Glu Thr 35 Gly ArgGin ‘Leu Leu 40 Cys Asp Lys Cys Ala 45 For Gly Thr Tyr Leu 50 Lys Gin His Cys Thr 55 Wall Arg Arg Lys Thr 60 Leu Cys Wall For Cys 65 For Asp Tyr Ser Tyr 70 Thr Asp Ser Trp His 75 Thr Ser Asp Glu Cys 80 Wall Tyr Cys Ser For 85 Wall Cys Lys Glu Leu 90 Gin Thr Wall Lys Gin 95 Glu

01-1718-97 č

218

Cys Asn Arg Thr 100 ALIGN! His Asn Arg Wall Cys 105 Glu Cys Glu Glu Gly 110 Arg Tyr Leu Glu Leu Glu Phe Cys Leu Lys His Arg Ser Cye For For Gly Leu 115 12 0 125 Gly Wall 130 Leu Gin Ala Gly Thr 135 For Glu Arg Asn Thr 140 Wall Cys Lys Arg Cys 145 For Asp Gly Phe Phe 150 Ser Gly Glu Thr Ser 155 Ser Lys Ala For Cys 160 Arg Lys His Thr Asn 165 Cys Ser Ser Leu Gly 170 Leu Leu Leu Ile Gin 175 Lys Gly Asn Ala Thr 180 H3 Asp Asn Wall Cys 185 Ser Gly Asn Arg Glu 190 Ala Thr Gin Asn Cys 195 Gly Ile Asp Wall Thr 200 Leu Cys Glu Glu Ala 205 Phe Phe Arg

Data of SEQ ID NO: 134: (and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 224 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:

Met Gly Ala Gly Ala Thr Gly Arg Ala Met Ašp Gly For Arg Lěu Leu 1 5 10 15 Dec Leu Leu Leu Leu Leu Gly Wall Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 May 25 30 For Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45 Leu Gly Glu Gly Wall Ala Gin For Cys Gly Ala Asn Gin Thr Wall Cys 50 55 60

··

01-1718-97 Če

219 • β * 9 4

9 «·· 99

Glu 65 For Cys Leu Asp Ser 70 Wall Thr Phe Ser Asp 75 Wall Wall Ser Ala Thr 80 Glu For Cys Lys For 85 Cys Thr Glu Cys Wall 90 Gly Leu Gin Ser Met 95 Ser Ala For Cys Wall 100 ALIGN! Glu Ala Asp Asp Ala 105 Wall Cys Arg Cys Ala 110 Tyr Gly Tyr Tyr Gin 115 Asp Glu Thr Thr Gly 120 Arg Cys Glu Ala Cys 125 Arg Wall Cys Glu Ala 130 Gly Ser Gly Leu Wall 135 Phe Ser Cys Gin Asp 140 Lys Gin Asn Thr Wall 145 Cys Glu Glu Cys For 150 Asp Gly Thr Tyr Ser 155 Asp Glu Ala Asn His 160 Wall Asp For Cys Leu 165 For Cys Thr Wall Cys 170 Glu Asp Thr Glu Arg 175 Gin Leu Arg Glu Cys 180 Thr Arg Trp Ala Asp 185 Ala Glu Cys Glu Glu 190 Ile For Gly Arg Trp 195 Ile Thr Arg Ser Thr 200 For For Glu Gly Ser 205 Asp Ser Thr Ala For 210 Ser Thr Gin Glu For 215 Glu Ala For For Glu 220 Gin Asp Leu Ile

(1) Data on SEQ ID NO: 135:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 205 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single

(D) TOPOLOGY: linear '(ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:

Met Thr Val Trp Val Gin Gin Pro Thr Ala Phe Leu Leu Leu Gly Leu 1 5 10 15

Ser Leu Gly Val Thr Val Lys Leu Asn Cys Val Lys Asp Thr Tyr Pro 20 25 30 ·· ··

01-1718-97 English ····

220

Ser Gly His 35 Lys Cys Cys Arg Glu 40 Cys Gin For Gly His 45 Gly Met Wall Ser Arg 50 Cys Asp His Thr Arg 55 Asp Thr Wall Cys His 60 For Cys Glu For Gly 65 Phe Tyr Asn Glu Ala 70 Wall Asn Tyr Asp Thr 75 Cys Lys Gin Cys Thr 80 Gin Cys Asn His Arg Ser Gly Ser Glu Leu Lys Gin Asn Cys Thr For 85 90 95 Thr Glu Asp Thr Wall Cye Gin Cys Arg For Gly Thr Gin For Arg Gin 100 ALIGN! 105 110 Asp Ser Ser 115 His Lys Leu Gly Wall 120 Asp Cys Wall For Cys 125 For For Gly

His Phe 130 Ser For Gly Ser Asn 135 Gin Ala Cys Lys For 140 Trp Thr Asn Cys Thr 145 Leu Ser Gly Lys Gin 150 Ile Arg His For Ala 155 Ser Asn Ser Leu Asp 160 Thr Wall Cys Glu Asp Arg Ser Leu Leu Ala Thr Leu Leu Trp Glu Thr 165 170 175 Gin Arg Thr Thr Phe Arg For Thr Thr Wall For Ser Thr Thr Wall Trp 180 185 190 For Arg Thr Ser Gin Leu For Ser Thr For Thr Leu Wall

195 200 205

Met (1) data for SEQ ID NO: 136:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 191 amino acids' ⁼ (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:

Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val 5 10 15 ·

01-1718-97 Če

221

Gly Cys Glu Lys Val Gly Ala Val Gin Asn Ser

For Gly Thr Phe Cys Arg Lye Tyr Asn For Val Cys Lys Ser Cys Pro 35 ..... 40 ...... · ’- 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Pro Asn Cys Asn Ile Cys 50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Ser Ser Thr

70

His Asn Ala Glu Cys Glu Cys

90 95

Gin Gys Glu Glu Glu Glu Glu Glu Glu Glu Glu Leu Thr 100 105 110

Lys Gin Gly Cys Lys Thr Cys Ser Leu Phly Asn Asp Gin Asn 115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys

Ser Val Leu Ly3 Thr Thr Thr Thr Thr Glu Lys Asp

145 150 155 160

Pro Val Val Ser Phe Ser Pro Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Gly Gly His Ser Leu Gin Val Leu Thr Leu Phe Leu 180,185,190 (1) Data on SEQ ID NO: 137:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C, CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:

TATGGATGAA GAAACTTCTC ATCAGCTGCT GTGTGATAAA TGTCCGCCGG GTAC

01-1718-97 Če

222 (1) data of SEQ ID NO: 138:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 380 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:

Glu 1 Thr Leu For For 5 Lys Tyr Leu His Tyr 10 Asp For Glu Thr Gly 15 Dec His Gin Leu Leu Cys 20 May Asp Lys Cys Ala For 25 Gly Thr Tyr Leu Lys 30 Gin His Cys Thr Wall 35 Arg Arg Lys Thr Leu 40 Cys Wall For Cys For 45 Asp His Ser Tyr Thr 50 Asp Ser Trp His Thr 55 Ser Asp Glu Cys Wall 60 Tyr Cys Ser For Wall 65 Cys Lys Glu Leu Gin 70 Ser Wall Lys Gin Glu 75 Cys Asn Arg Thr His 80 Asn Arg Wall Cys Glu 85 Cys Glu Glu Gly Arg 90 Tyr Leu Glu Ile Glu 95 Phe Cys Leu Lys His 100 ALIGN! Arg Ser Cys For For 105 Gly Ser Gly Wall Wall 110 Gin Ala Gly Thr For 115 Glu Arg Asn Thr Wall 120 Cys Lys Lys Cys For 125 Asp Gly Phe _Phe Ser 130 Gly Glu ~ : Thr Ser Ser 135 Lys Ala Pro = Cys Ile ’ 140 Lys “His Thr Asn Cys 145 Ser Thr Phe Gly Leu 150 Leu Leu Ile Gin Lys 155 Gly Asn Ala Thr His 160 Asp Asn Wall Cys Ser 165 Gly Asn Arg Glu Ala 170 Thr Gin Lys Cys Gly 175 Ile Asp Wall Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Wall For Thr

180 185 190

01-1718-97 Čé

223

Lys Ile Ile Pro Asn 195 Trp Leu Ser Val Leu Gly 200 205 Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg Ile Lys Arg Arg H3 Ser 210 215 220 Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn 225 230 235 240 Arg Asp Gin Glu Met Wall Lys Lys Ile Ile Gin Aep Ile Asp Leu Cys 245 250 255 Glu Ser Ser Wall Gin Arg His Leu Gly His Ser Asn Leu Thr Thr Glu 260 265 270 Gin Leu Leu Ala Leu Met Glu Ser Leu For Gly Lys Lys Ile Ser For 275 280 285 Glu Glu Ile Glu Arg Thr Arg Lys Thr Cys Lys Ser Ser Glu Gin Leu 290 295 300 Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gin Asp Thr 305 310 315 320 Leu Lys Gly Leu Met Tyr Ala Leu Lys His Leu Lys Thr Ser His Phe 325 330 335 For Lys Thr Wall Thr His Ser Leu Arg Lys Thr Met Arg Phe Leu His 340 345 350 Ser Phe Thr Met Tyr Arg Leu Tyr Gin Lys Leu Phe Leu Glu Met Ile 355 360 365 Gly Asn Gin Wall Gin Ser Wall Lys Ile Ser Cys Leu 370 375 380

(1) Data on SEQ ID NO: 139:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 380 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein

01-1718-97 Č

224 (xi) SEQ ID NO: 139:

Glu Thr Phe 2 For For Lys 5 Tyr Leu His Tyr Asp Glu Glu _.......... 10 ...... Thr Ser 15 Dec His Gin Leu Leu Cys Asp Lys Cys For For Gly Thr Tyr Leu Lys Gin Hlá 20 May 25 30 and Cys Thr Ala Lys Trp Lys Thr Wall Cys Ala For Cys For Asp His Tyr 35 40 45 Tyr Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser For 50 55 60 Wall Cys Lys Glu Leu Gin Tyr Wall Lys Gin Glu Cys Asn Arg Thr His 65 70 75 80 Asn Arg Wall Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Ile Glu Phe 85 90 95 Cys Leu Lys His Arg Ser Cy3 For For Gly Phe Gly Wall Wall Gin Ala 100 ALIGN! 105 110 Gly Thr For Glu Arg Asn Thr Wall Cys Lys Arg Cys For Asp Gly Phe 115 120 125 Phe Ser Asn Glu Thr Ser Ser Lys Ala For Cys Arg Lys His Thr Asn 130 135 140 Cys Ser Wall Phe Gly Leu Leu Leu Thr Gin Lys Gly Asn Ala Thr His 145 150 155 160 Asp Asn Ile Cys Ser Gly Asn Ser Glu Ser Thr Gin Lys Cys Gly Ile 165 170 175 Asp Wall Thr Leu Cys Glu Glu Ala Phe Phe Arg Phe Ala Wall For Thr 180 185 190 Lys Phe Thr For Asn Trp Leu Ser Wall Leu Wall Asp Asn Leu For Gly 195 200 = _______ ... • · 205 —- Thr Lys Wall Asn Ala Glu Ser Wall Glu Arg Ile Lys Arg Gin His Ser 210 215 220 Ser Gin Glu Gin Thr Phe Gin Leu Leu Lys Leu Trp Lys His Gin Asn

(+420) 225 230 235 240

Lys Ala Gin Asp Ile Val Lys Ile Ile Gin Asp Ile Asp Leu Cys 245 250 255 ·· • · ς

• · ····

01-1718-97 Ce ···

225 ·························

Glu Asn Ser Wall 260 Gin Arg His Ile Gly His Ala Asn Leu Thr Phe Glu 265 270 Gin Leu Arg Ser Leu Met Glu Ser Leu For Gly Lys Lys Wall Gly Ala 275 280 ²⁸⁵ Glu Asp Ile Glu Lys Thr Ile Lys Ala Cys Lys For Ser Asp Gin Ile 290 295 300 Leu Lys Leu Leu Ser Leu Trp Arg Ile Lys Asn Gly Asp Gin Asp Thr 305 310 315 320 Leu Lys Gly Leu Met His Ala Leu Lys His Ser Lys Thr Tyr His Phe 325 330 335 For Lys Thr Wall Thr Gin Ser Leu Lys Lys Thr Ile Arg Phe Leu His 340 345 350 Ser Phe Thr Met Tyr Lys Leu Tyr Gin Lys Leu Phe Leu Glu Met Ile 355 360 365 Gly Asn Gin Wall Gin Ser Wall Lys Ile Ser Cys Leu 370 375 380 (1) detail e k SEQ NO ID: 140:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:

TGGACCACCC AGAAGTACCT TCATTATGAC 30 (1) data for SEQ ID NO: 141:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear ·· o ·

01-1718-97 Če

226

(ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:

GTCATAATGA AGGTACTTCT GGGTGGTCCA '' 30 (1) data for SEQ ID NO: 142:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142:

GGACCACCCA GCTTCATTAT GACGAAGAAA C 31 (1) data for SEQ ID NO: 143:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:

GTTTCTTCGT CATAATGAAG CTGGGTGGTC C 31 (1) data for SEQ ID NO: 144:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

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I *

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227 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144:

GTGGACCACC CAGGACGAAG AAACCTCTC ..... 29 ‘

From (1) the data for SEQ ID NO: 145:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:

GAGAGGTTTC TTCGTCCTGG GTGGTCCAC 29 (1) data for SEQ ID NO: 146:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146:

CGTTTCCTCC AAAGTTCCTT CATTATGAC 29 (1) data for SEQ ID NO: 147:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

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228

• ·

(Ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147:

GTCATAATGA AGGAACTTTG GAGGAAACG .....-- - ₂ 9 (1) data for SEQ ID NO: 148:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148:

GGAAACGTTT CCTGCAAAGT ACCTTCATTA TG 32 (1) data for SEQ ID NO: 149:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149:

CATAATGAAG GTACTTTGCA GGAAACGTTT CC 32 (1) data for SEQ ID NO: 150:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear • ·

01-1718-97 Če

(Ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:

CACGCAAAAG TCGGGAATAG ATGTCAC ~ ¹ 27 (1) data of SEQ ID NO: 151:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151:

GTGACATCTA TTCCCGACTT TTGCGTG 27

data for SEQ ID NO: 152: (and) CHARACTERISTIC SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) SPECIES MOLECULES: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152

CACCCTGTCG GAAGAGGCCT TCTTC 25 (1) data for SEQ ID NO: 153:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

01-1718-97 Če

(Ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:

'GAAGAAGGCC TCTTCCGACA GGGTG''- ----- - - -' - - - '- - - - - - ₂ 5 /

(1) Data on SEQ ID NO: 154:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154:

TGACCTCTCG GAAAGCAGCG TGCA 24 (1) data for SEQ ID NO: 155:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:

TGCACGCTGC TTTCCGAGAG GTCA 24 (1) data for SEQ ID NO: 156:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

01-1718-97 Če

231 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:

CCTCGAAATC GAGCGAGCAG CTCC 24 (1) data for SEQ ID NO: 157:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:

CGATTTCGAG GTCTTTCTCG TTCTC 25

(1) entry e to SEQ ID NO: 158: (and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158 CCGTGAAAAI 'AAGCTCGTTA TAACTAGGAA (1) entry e to SEQ ID NO: 159: (and) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear

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01-1718-97 English ····

232 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:

CCATTCCTAG TTATAACGAG CTTATTTTCA CGG '33' (1) Data to SEQ ID NO: 160:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:

CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (1) data for SEQ ID NO: 161:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:

CCTCTCTCGAGTCAGGTGAC ATCTATTCCA CACTTTTGCG TGGC 44 (1) data for SEQ ID NO: 162:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear ·· · · · · ·

* ···

233 (ii) MOLECULAR TYPE: cDNA

(17) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 162:

CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG (1) data for SEQ ID NO: 163:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:

CCTCTCTCGA GTCAAGGAAC AGCAAACCTG AAGAAGGC 38 (1) data for SEQ ID NO: 164:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:

CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (1) data for SEQ ID NO.-165:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear

234

01-1718-97 (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:

CCTCTCTCGA GTCACTCTGT GGTGAGGTTC GAGTGGCC "38 .......!

(1) Data on SEQ ID NO: 166:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:

CCTCTGAGCT CAAGCTTCCG AGGACCACAA TGAACAAG 38 (1) data for SEQ ID NO: 167:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:

CCTCTCTCGA

GTCAGGATGT TTTCAAGTGC TTGAGGGC (1) data for SEQ ID NO: 168:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: single (D) TOPOLOGY: linear ··

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99

235 (ii) MOLECULAR TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:

Met Lys His His His His' Ala Ser Val Asn Ala Leu Glu

Claims (51)

PATENT CLAIMS An isolated nucleic acid encoding a polypeptide comprising at least one of the biological activities of OPG, characterized in that it is selected from the group consisting of: (a) the nucleic acids shown in Figures 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122) and 9C to 9D (SEQ ID NO: 124), or their complementary strands; b) nucleic acids that hybridize under stringent conditions to the polypeptide coding regions depicted in Figures 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122), and 9C to 9D (SEQ ID NO: 124); (c) nucleic acids which hybridize under stringent conditions to nucleotides 148 to 337 inclusive, depicted in Figure IA; and d) nucleic acids which are degenerates of nucleic acids (a), (b) and (c). 2. Nucleic acid according to claim 1, characterized in that it is cDNA, genomic DNA, synthetic DNA or RNA. ..... - '’..... The polypeptide encoded by the nucleic acid of claim 1. • · 01-1718-97 Če 237 4. Nukleová acid according to claim 1, v y z n a. č e - on by that includes one : or several codons, conveniently for expression of Escherichia coli. • 5. Nukleová acid according to claim 1, v y z n a č e - by having a detectable label attached thereto. 6. The nucleic acid of claim 1, comprising the polypeptide-coding region of FIGS. 2B to 2C (SEQ ID NO: 120), 9A to 9B (SEQ ID NO: 122) and 9C to 9D (SEQ ID NO: 124). ). Nucleic acid according to claim 6, characterized in that it has the sequence shown in Figures 9C to 9D (SEQ ID NO; 124) of nucleotides 158 to 1297. An expression vector comprising the nucleic acid of claim 1. The expression vector of claim 8, wherein the nucleic acid comprises the polypeptide-coding region shown in Figures 9C-9D (SEQ ID NO: 124). ·· 01-1718-97 Če 238 A host cell transformed or transfected with an expression vector according to claim 8. 11. A host cell according to claim 10, characterized in that it is a eukaryotic cell. 12. The host cell of claim 11, wherein the cell is selected from the group consisting of: from the group comprising! CHO, COS, 293, 3T3, CV-1 and BHK cells. 13. A host cell according to claim 10, characterized in that it is a prokaryotic cell. A host cell according to claim 13, characterized in that it is Escherichia coli. A transgenic mammal comprising the expression vector of claim 8. 16. The transgenic mammal of claim 15, wherein the mammal is a rodent. · ftft ftftft · ftftft · ft »» * ft * _in ··· · · ft ft ft ftftft · · 01-1718-97 ce / ftftftft ftftft ftftft 239 17. The transgenic mammal of claim 16, wherein the mammal is a mouse. and 18. A method for producing OPG comprising: growing host cells transformed or transfected with the nucleic acid of claim 1 under appropriate nutrient conditions; and isolating the polypeptide products of the nucleic acid expression. OPG. A purified and isolated polypeptide comprising The polypeptide of claim 19 that is mammalian OPG. mark by The polypeptide of claim 20 that is human OPG. marked 22. A polypeptide by being a protein. according to the claim substantially 19, in plain marked other human by 23. A polypeptide according to claim 21 having the amino acid sequence shown in FIG. 9A to (SEQ ID NO: 125) labeled in FIG. 01-1718-97 Če 240 of Figures 2B to 2C (SEQ ID NO: 121), 9B (SEQ ID NO: 123) or Figures 9C to 9D or derivatives thereof. 24. The polypeptide of claim 23 having the amino acid sequence of FIGS. 9C to 9D (SEQ ID NO: 125) consisting of residues 22-401 inclusive. 25. The polypeptide of claim 23 having the amino acid sequence shown in FIGS. 9C to 9D (SEQ ID NO: 125) consisting of residues 32-401 inclusive. 26. The polypeptide of claim 19, wherein the polypeptide is an expression product of an exogenous DNA sequence. 27. The polypeptide of claim 26, wherein the DNA is cDNA, genomic DNA or synthetic DNA. _......, 28. The polypeptide of claim 19, wherein the polypeptide is modified with a water-soluble polymer. 01-1718-97 Če 241 29. The polypeptide of claim 28, wherein the water-soluble polymer is polyethylene glycol. A polypeptide comprising: an amino acid sequence of at least 164 amino acids comprising four cysteine rich domains characterized by cysteine rich domains of the extracellular regions of tumor necrosis factor factor receptors; and bone density increasing activity. A polypeptide comprising the amino acid sequence shown in Figure 2B to 2C (SEQ ID NO: 121), Figure 9A to 9B (SEQ ID NO: 123), or Figure 9C to 9D (SEQ ID NO: 125) having a 22 N residue and wherein acids 1 to 216 are omitted from the carboxyl terminus. 32. The polypeptide of claim 31, comprising the amino acid sequence of residues 22-185, 22-189, 22-194, or 22-201 inclusive. 33. The polypeptide of claim 32, further comprising a human IgG1 Fc region extending from the carboxyl terminus. 34. A polypeptide comprising the amino acid sequence shown in Figure 2B to 2C (SEQ ID NO: 121), Figure 9A. 01-1718-97 Che · j *. 242 to 9B (SEQ ID NO: 123) or Figure 9C to 9D (SEQ ID NO: 125) having an N-terminus at residue 22 and wherein 1 to 10 amino acids are omitted at the N-terminus and optionally at the carboxyl terminus 1 up to 216 acids. 35. The polypeptide of claim 34, comprising the amino acid sequence of residues 27-185, 27-189, 27-401, or 32-401 inclusive. 36. The polypeptide of claim 35, further comprising a human IgG1 Fc region extending from the carboxyl terminus. 37. A polypeptide selected from the group consisting of: huOPG [22-201] -Fc huOPG [22-401] -Fc huOPG [22- 180] -Fc huOPG met [22-401] -Fc huOPG Fc-met [22-401] huOPG met [22-185] huOPG met [22-189] huOPG met [22-194] huOPG met [27-185] huOPG met [27-189] huOPG met [27-194] huOPG met [32-401] huOPG met- Lys [22-401] huOPG met [22-401] huOPG met [22-401] -Fc P25A • · ·· * . · ·· ·· · 01-1718-97 Che  ..... - - huOPG met [22-401] 243 (P25A) huOPG met [22-401] (P2 6A) huOPG met [22-401] (P26D) huOPG ’ met [22-194] (P25A) huOPG met [22-194] (P26A) huOPG met met- (lys) ₃ [22-401] huOPG met Met-arg-gly-ser- (his) ₆ [22-401]. 38. A nucleic acid encoding the polypeptide of claim 37. 39. An antibody or fragment thereof that specifically binds to OPG. 40. The antibody of claim 39, wherein the antibody is a monoclonal antibody. 41. A method for detecting the presence of OPG in a biological sample, comprising: incubating the sample with the antibody of claim 39 for. conditions that allow binding of the antibody to OPG; and detecting the bound antibody. 42. A method for assessing the ability of a candidate substance to bind to an OPG, comprising: 01-1718-97 9 • Β β * * Φ * · W 9 9 · 99 9 · 244 incubating the OPG with the candidate agent under conditions that allow its binding; and measuring the bound substance. 43. A method for controlling OPG levels in an animal body comprising modifying the animal with a nucleic acid encoding OPG. 44. The method of claim 43, wherein the nucleic acid promotes an increase in OPG levels in the tissue. 45. The method of claim 44, wherein the animal is a human. 46. A pharmaceutical composition comprising a therapeutically effective amount of OPG in a pharmaceutically acceptable carrier, adjuvant, solubilizing agent, stabilizing agent and / or antioxidant. 47. team The composition of claim 46, wherein the OPG is human OPG. marked with \ X 01, 1718-97 Ce, J. J. * 245 48. The composition of claim 47, wherein the OPG has the amino acid sequence shown in Figure 9B. . . . . . , ... / 49. A method of treating bone disease comprising administering a therapeutically effective amount of the polypeptide of claim 19. 50. The method of claim 49, wherein the polypeptide is human OPG. 51. The method of claim 49, wherein the bone disease is excessive bone loss. 52. The method of claim 51, wherein the bone disease is selected from the group consisting of osteoporosis, Paget's bone disease, hypercalcemia, hyperparathyroidism, steroid-induced osteopenia, bone loss due to joint rheumatism, bone loss due to osteomyelitis, osteolytic metastasis, and osteolytic metastasis. periodontal bone loss. 53. The method of claim 49, further comprising administering a therapeutically effective amount of a substance selected from the group consisting of bone morphogenic proteins BMP-1 to BMP-12, members of the TGF-β family, aa and a and e and a. ·· 01-1718-97 Che 'aa aa aa aa 246 IL-1 inhibitors, TNFα inhibitors, parathyroid hormone-derived protein and analogs thereof, E series prostaglandins, bisphosphonates and bone-enriching minerals. 54. Osteoprotegerin multimer consisting of osteoprotegerin monomers. 55. Osteoprotegerin multimer by claim 54, v y marked by tim, that means dimer. 56. Osteoprotegerin multimer by claim 54, v y marked by tim, that it consists of an internal chain disulfide bonds. 57. Osteoprotegerin multimer according to claim 54, marked by that is formed by an Fc linkage sections derived from human IgG1. 58. Osteoprotegerin multimer ” according to claim 54, characterized in that it is substantially free of osteoprotegerin monomers and inactive multimers. 59. The osteoprotegerin multimer of claim 54 wherein the monomers comprise the amino acid sequence shown in FIGS. 9C-9D. 9 - 01-1718-97 9 »· i 9 6 · 9 • 247 (SEQ ID NO: 125) consisting of residues 22-401, or a derivative thereof. 60. The osteoprotegerin multimer of claim 54, wherein the monomers comprise the amino acid sequence shown in FIGS. 9C-9D (SEQ ID NO: 125) consisting of residues 22-194, or a derivative thereof.